Funcionalidad del aceite de oliva virgen extra en la artritis reumatoide experimental

DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA FUNCIONALIDAD DEL ACEITE DE OLIVA VIRGEN EXTRA EN LA ARTRITIS REUMATOIDE EXPERIMENTAL Tesis Doctoral presentada por MARÍA DE LOS ÁNGELES ROSILLO RAMÍREZ Para optar al Grado de Doctora en Farmacia Sevilla, 2015 tu4o - lD¡ o fll DEPARTAMENTo Df, FAnMACoLociA FACULTAD DE FARMACIA UNTVERSIDAD DE SEVILLA Las Dras. Catalina Alarcón de la Lastra Romero, Catedrática de Universidad y Marina Sánchez Hidalgo, Profesora Contratada Doctora, adscritas al Departamento de Farmacología de la Facultad de Farmacia de la Universidad de Sevilla. IN FORMAN QUE IA TCSiS DOCIOTAI titUIAdA "FUNCIONALIDAD DEt ACEITE DE OLIVA VIRGEN EXTRA EN LA ARRITIS REUMATOIDE EXPERIMENTAL" presentada por la Lda. María de los Ángeles Rosillo Ramírez para optar al grado de Doctora en Farmacia con Mención lnternacional ha sido llevada a cabo bajo su dirección. Sevilla, 16 de noviembre de 2015 I (rjf-'¿J' L4 /¿-\ Dra. Catalina Alarcón de la Lastra Romero Dra. Marina Sánchez Hidalgo tD4oó m DEPARTAMENTO DE FARMACOLOGiA FACULTAD DE FARMACIA I]N T!'ERSID{) DE SDI'ILLA Dña. ME Dolores García Giménez, Profesora Catedrát¡ca de Farmacología y Directora del Departamento de Farmacología de Ia Facultad de Farmacia de la Universidad de Sevilla. CERTIFICA: Que la Tesis Doctoral titulada "FUNCIONALIDAD DEL ACEITE DE OIIVA VIRGEN EXTRA EN tA ARRITIS REUMATOIDE EXPERIMENTAL" realizada por la Lda. María de los Ángeles Rosillo Ramírez, ha sido dirigida por la Dra. Catal¡na Alarcón de la Lastra Romero y la Dra. Mar¡na Sánchez Hidalgo, para aspirar al grado de Doctor en Farmacia con Mención lnternac¡onal, cumpliendo los requisitos para este tipo de trabajo. Y para que asíconste, firmo la presente. En Sevilla, a 16 de noviembre de 2013 Dra. Me Dolores García Este trabajo de investigación se ha llevado a cabo gracias a la financiación de las siguientes instituciones y proyecto de investigación: PROYECTO DE INVESTIGACIÓN ‒ Valoración del Aceite de Oliva Virgen Extra en la Artritis Reumatoide Experimental: Estudio Biodirigido, Caracterización Farmacológica y Desarrollo de Ingredientes Funcionales. Entidad financiadora: Consejería de Innovación, Ciencia y Empresa. Junta de Andalucía. Investigador principal: Catalina Alarcón de la Lastra Romero. Referencia: AGR-6609 Duración: 2011- 2016 BECA PREDOCTORAL Beca predoctoral asociada a Proyectos de Investigación de Excelencia en Equipos de Investigación Orden de 11 de Diciembre de 2007 (BOJA 4, 05/01/2008) Convocatoria 2010 (BOJA 135, 12/07/2011). Entidad financiadora: Consejería de Innovación, Ciencia y Empresa. Junta de Andalucía. Y gracias a las estancias realizadas en: ‒ Lugar: Departamento de Farmacología. Facultad de Farmacia. Universidad de Valencia, España. Duración: Octubre-Noviembre 2012. Investigador responsable: María José Alcaraz Tormo. ‒ Lugar: Departamento de Reumatología. Erasmus MC. Universidad Erasmus. Rotterdam, Holanda Duración: Septiembre-Diciembre 2014. Investigador responsable: Erik Lubberts. Publicaciones María de los Ángeles Rosillo Ramírez, Licenciada en Farmacia por la Universidad de Sevilla en 2009. En 2010 cursó el Máster en Atención Farmacéutica y Farmacia Asistencial en la misma universidad, durante este año recibió una beca de Plan de Apoyo al Máster Oficial de la Universidad de Sevilla. Ese mismo año consiguió el Premio a la mejor Beca sobre “Tema Libre” “Actividad del zumo de la granada y sus componentes en la inflamación crónica intestinal asociada a cáncer colorrectal experimental” concedido por la Fundación Farmacéutica Avenzoar. Desde el año 2010 forma parte del grupo de investigación Farmacología Experimental y Farmacia Clínica (CTS-259). Comenzó sus estudios de Doctorado en la Universidad de Sevilla en el año 2011. En 2012 obtuvo una beca predoctoral Adscrita a Proyectos de Investigación de Excelencia en Equipos de Investigación asociada al proyecto AGR 6609 de la Consejería de Economía, Innovación y Ciencia de la Junta de Andalucía para la realización de su Tesis Doctoral bajo la dirección de las doctoras Catalina Alarcón de la Lastra Romero y Marina Sánchez Hidalgo. Completó su formación predoctoral en el Departamento de Farmacología de la Universidad de Valencia bajo la dirección de la doctora Mª José Alcaraz Tormo durante los meses de octubre y noviembre de 2012 y en el Departamento de Reumatología e Inmunología del Erasmus Medical Center de Rotterdam (Holanda) bajo la dirección del doctor Erik Lubberts durante los meses de septiembre a diciembre de 2014. Ha participado en distintos proyectos, así como en diversos trabajos de investigación que han sido presentados en congresos nacionales e internacionales y que han dado lugar a distintas publicaciones en revistas internacionales. PROYECTOS DE INVESTIGACIÓN ‒ Valoración del Aceite de Oliva Virgen Extra en la Artritis Reumatoide Experimental: Estudio Biodirigido, Caracterización Farmacológica y Desarrollo de Ingredientes Funcionales (AGR-6609 – Becaria predoctoral) Junta de Andalucía. Investigador principal: Dra. Catalina Alarcón de la Lastra Romero. Publicaciones PUBLICACIONES EN REVISTAS INTERNACIONALES Derivadas de la tesis: ‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fernández-Bolaños JG, Lubberts E, Alarcón-de-la-Lastra C. Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis. Mol Nutr Food 2015. In press. ‒ Rosillo MA, Sánchez-Hidalgo M, Sánchez-Fidalgo S, Aparicio-Soto M, Villegas I, Alarcónde-la-Lastra C. Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen-induced arthritis. Eur J Nutr. 2015. In press. ‒ Rosillo MA, Alcaraz MJ, Sánchez-Hidalgo M, Fernández-Bolaños JG, Alarcón-de-la-Lastra C, Ferrándiz ML. Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis. J Nutr Biochem. 2014;25(12):1275-81. No relacionadas con la tesis: ‒ Aparicio-Soto M, Sánchez-Hidalgo M, Cárdeno A, Rosillo MA, Sánchez-Fidalgo S, Utrilla J, Martín-Lacave I, Alarcón-de-la-Lastra C. Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf-2 antioxidant pathway and suppression of JAK/STAT, NF-κB and MAPK activation. J Nutr Biochem. 2015. In press. ‒ Sánchez-Fidalgo S, Villegas I, Aparicio-Soto M, Cárdeno A, Rosillo MA, González- Benjumea A, Marset A, López Ó, Maya I, Fernández-Bolaños JG, Alarcón de la Lastra C. Effects of dietary virgin olive oil polyphenols: hydroxytyrosyl acetate and 3, 4- dihydroxyphenylglycol on DSS-induced acute colitis in mice. J Nutr Biochem. 2015;26(5):513-20. ‒ Sánchez-Fidalgo S, Villegas I, Rosillo MA, Aparicio-Soto M, de la Lastra CA. Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways. Mol Nutr Food Res. 2015;59(2):284-92. Publicaciones ‒ González-Mauraza H, Martín-Cordero C, Alarcón-de-la-Lastra C, Rosillo MA, León- González AJ, Sánchez-Hidalgo M. Anti-inflammatory effects of Retama monosperma in acute ulcerative colitis in rats. J Physiol Biochem. 2014;70(1):163-72. ‒ Cárdeno A, Sánchez-Hidalgo M, Rosillo MA, Alarcón de la Lastra C. Oleuropein, a secoiridoid derived from olive tree, inhibits the proliferation of human colorectal cancer cell through downregulation of HIF-1α. Nutr Cancer. 2013;65(1):147-56. ‒ Sánchez-Fidalgo S, Cárdeno A, Sánchez-Hidalgo M, Aparicio-Soto M, Villegas I, Rosillo MA, de la Lastra CA. Dietary unsaponifiable fraction from extra virgin olive oil supplementation attenuates acute ulcerative colitis in mice. Eur J Pharm Sci. 2013;48(3):572-81. ‒ Rosillo MA, Sánchez-Hidalgo M, Cárdeno A, Aparicio-Soto M, Sánchez-Fidalgo S, Villegas I, de la Lastra CA. Dietary supplementation of an ellagic acid-enriched pomegranate extract attenuates chronic colonic inflammation in rats. Pharmacol Res. 2012;66(3):235- 42. ‒ Rosillo MA, Sánchez-Hidalgo M, Cárdeno A, de la Lastra CA. Protective effect of ellagic acid, a natural polyphenolic compound, in a murine model of Crohn's disease. Biochem Pharmacol. 2011;82(7):737-45. COMUNICACIONES A CONGRESOS Derivadas de la tesis: Internacionales ‒ Rosillo MA, Alcaráz MJ, Ferrándiz, ML, Sánchez-Hidalgo M, Sánchez-Fidalgo S, Villegas I, Alarcón-de-la-Lastra C. “POLYPHENOL EXTRACT FROM EXTRA VIRGIN OLIVE OIL AMELIORATES TYPE II COLLAGEN-INDUCED ARTHRITIS IN DBA/1 J MICE” Póster. 13th ISANH International Conference on Antioxidants. Marrakech (Morocco), Junio de 2013. Nacionales Publicaciones ‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fernández-Bolaños JG, Méndez- Gutiérrez A, Pérez-Domíngez M, Villegas I, Lubberts E, Alarcón-de-la-Lastra C. DIETARY HYDROXYTYROSOL ACETATE PREVENTS INFLAMMATORY RESPONSE AND JOINT DAMAGE IN MURINE EXPERIMENTAL ARTHRITIS. Comunicación oral premiada. 36 Congreso de la Sociedad Española de Farmacología. Valencia, Septiembre de 2015. ‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fernández-Bolaños JG, Méndez- Gutiérrez A, Pérez-Domíngez M, Villegas I, Lubberts E, Alarcón-de-la-Lastra C. ANTIINFLAMMATORY EFFECTS AND JOINT PROTECTION OF A HYDROXYTYROSOL ACETATE ENRICHED DIET, AN EXTRA VIRGIN OLIVE OIL POLYPHENOL, IN COLLAGEN-INDUCED ARTHRITIS IN MICE. Comunicación oral. VII Jornadas de Jóvenes Farmacólogos de Andalucía. Granada, Julio de 2015. ‒ Rosillo MA, Ferrándiz ML, Sánchez-HidalgoML, Fernández-Bolaños JG, Alarcón-de-la- Lastra C, Alcaraz MJ. ANTI-INFLAMMATORY AND JOINT PROTECTIVE EFFECTS OF A POLYPHENOLIC EXTRACT FROM EXTRA VIRGIN OLIVE OIL IN TYPE II COLLAGEN-INDUCED ARTHRITIS. Póster. XXXV Congreso de la Sociedad Española de Farmacología. Madrid, Septiembre de 2014. ‒ Rosillo MA, Méndez-Gutiérrez A, Ferrandiz ML, Sánchez-Hidalgo M, Fernández-Bolaños JG, Alarcón-de-la-Lastra C, Alcaraz MJ. ANTI-IFLAMMATORY AND JOINT PROTECTIVE EFFECTS OF EXTRA VIRGIN OLIVE OIL POLYPHENOL EXTRACT IN EXPERIMENTAL ARTHRITIS. Comunicación oral. VI Jornadas de Jóvenes Farmacólogos de Andalucía. Sevilla, Junio de 2014. No relacionados con la tesis: Internacionales ‒ Rosillo MA, Sánchez-Hidalgo M, Cárdeno A, Aparicio-Soto M, Conde M, Sánchez-Fidalgo S, Villegas I, Alarcón-de-la-Lastra C. BENEFICIAL EFFECTS OF AND ELLAGIC ACIDENRICHED POMEGRANATE EXTRACT ON CHRONIC TNBS-INDUCED COLITIS IN RATS. Póster. 6th European Congress of Pharmacology. Granada (Spain), Julio de 2012. ‒ Sánchez-Fidalgo S, Cárdeno A, Aparicio-Soto M, Villegas I, Sánchez-Hidalgo M, Rosillo MA, Alarcón-de-la-Lastra C. EFFECTS OF DIETARY UNSAPONIFIABLE FRACTION FROM Publicaciones EXTRA VIRGIN OLIVE OIL ON ACUTE ULCERATIVE COLITIS. Póster. 20th United European Gastroenterology Week (UEGW) Amsterdam, Octubre de 2012. GUT: BMJ; 2012. ‒ Sánchez-Fidalgo S, Cárdeno A, Aparicio-Soto M, Sánchez-Hidalgo M, Rosillo MA, Villegas I, Alarcón-de-la-Lastra C. DIETARY EXTRA VIRGIN OLIVE OIL POLPHENOLS SUPLEMENTATION MODULATES DSS-INDUCED CHRONIC COLITIS IN MICE. Póster. 20th United European Gastroenterology Week (UEGW) Amsterdam, Octubre de 2012. GUT: BMJ; 2012. ‒ Paras-Chavez C, Masoodi M, Sánchez-Hidalgo M, Noakes P, Alarcón-de-la-Lastra C, Rosillo MA. THE ROLE OF NULEAR FACTOR κβ ON THE SYNTHESIS OF LIPID INFLAMMATORY MEDIATORS ON THP-1 DERIVED MACROPHAGES. Póster. 10th Congress of the International Society for the Study of Fatty Acids and Lipids. Vancouver, 2012. ‒ Cárdeno A, Sánchez-Hidalgo M, Ramirez E, Aparicio-Soto M, Rosillo MA, Alarcón-de-la- Lastra C. NATURAL DIETARY POLYPHENOL, ELLAGIC ACID SUPRESSES COX-2 EXPRESSION IN HUMAN MONOCYTE THP-1 MACROPHAGES THROUGH MAPKs PATHWAYS. Póster. 19th United European Gastroenterology Week (UEGW). Stockholm, Octubre de 2011. GUT 2011; 60 (Suppl 3) A1. ‒ González-Mauraza NH, Sánchez-Hidalgo M, Martín-Cordero C, Rosillo MA, Talero E, Alarcón-de-la-Lastra C. RETAMA MONOSPERMA (L.) BOISSIER AQUEOUS EXTRACT ATTENUATES COLONIC INFLAMATION IN AN EXPERIMENTALLY ACUTE TNBS-INDUCED COLITIS MODEL. Comunicación oral. 18th United European Gastroenterology Week (UEGW) Barcelola, Octubre de 2010. GUT:BMJ; 2010. ‒ Rosillo MA, Sánchez-Hidalgo M, Cárdeno A, Alarcón-de-la-Lastra C. THE EFFECTS OF ELLAGIC ACID, A POLYPHENOL DERIVED FROM POMEGRANATE, IN A EXPERIMENTALLY ACUTE TNBS-INDUCED COLITIS MODEL. Póster. 18th United European Gastroenterology Week (UEGW) Barcelola, Octubre de 2010. GUT:BMJ; 2010. Nacionales Introducción 3 1. Rheumatoid Arthritis Rheumatoid arthritis (RA) can be defined as a chronic inflammatory disease with systemic autoimmune component, and is mainly characterized by aggressive synovial hyperplasia, synovitis, progressive destruction of cartilage, and bone erosion with painful swelling of small joints, fatigue, prolonged stiffness, and fever caused by immune responses and specific innate inflammatory processes (Zvaifler 1965, Creemers 2004, Brooks 2006). Global prevalence of RA has been estimated to be around 0.5-1.0% of adults in developed countries with a large variation across regions and approximately three-times more common in the female gender. The disease may begin at any age, but around 80% of all patients initiate the disease between the ages of 35 and 50 years (Rudan, Sidhu et al. 2015). RA patients exhibit an inflammatory chronic condition, which usually affects symmetrically diarthrodial and small joints of hands and feet. RA is characterized by the inflammation of synovial joint tissues leading hyperplasia of the synovial lining cell layer and the formation of rheumatoid pannus, which is capable of destroying adjacent cartilage and bone and causing subsequent joint deformity (Verpoort, van Dongen et al. 2004). Additional characteristic features of RA synovitis include neo-angiogenesis and infiltration of immune cells such as macrophages and lymphocytes which can form aggregates. Autoimmunity, identified by the production of auto-antibodies such as rheumatoid factor (RF) or anti-citrullinated protein antibodies (ACPA) precedes the clinically detectable onset of inflammatory arthritis and can last for years (Chapuy-Regaud, Nogueira et al. 2005, Wegner, Lundberg et al. 2010). The clinic outcome is extremely variable and may extend from a soft autolimitant AR to a multisystemic inflammation with a rapid outcome (Finckh 2009). In this sense, about 40% of patients, AR disease may involve an extraarticular component, affecting various organs and systems including skin, lungs, eyes, heart and blood vessels. The progressive articular or extra-articular deterioration, leads deformity, pain, functional disability and a decrease in life expectancy of Introducción 4 these patients. These characteristics justify its multidisciplinary approach, which includes medical treatment, physiotherapy and nutritional therapy. The different treatments are aimed primarily to suppress the inflammatory process alleviating the symptoms and signs of the disease. The etiology of RA is unknown. A genetic predisposition suggested by a link with HLADR4 and related allotypes of MHC class II, the T-cell-associated protein PTPN22, STAT4 and TRAFI1/C5, among others gene polymorphisms, has been related to a worse outcoming and a higher degree of articular destruction and extra-articular manifestations (Messemaker, Huizinga et al. 2015). However, the concordance rate of RA in monozygotic twins is low but higher than that in dizygotic twins suggesting that in addition to genetic factors, environmental factors as well as their interaction can be decisive in the development and disease progression. Among environmental factors, smoking has been recognized as a major risk factor RA. In fact, smoking has been associated with an increased risk of developing seropositive RA (RF and/or ACPA) (Chang, Yang et al. 2014). Nevertheless, other environmental factors including socio-economic, hormonal, silica exposure, stimuli like viruses, bacteria and stress, and dietetic factors may contribute to the disease progression (Cutolo 2007, Gomez-Puerta, Gedmintas et al. 2013). The pathogenesis of RA is incompletely understood but hyperplasia of the synovial membrane is a hallmark of RA pathology, which is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells, including CD4+ T- cells and innate immune cells (Komatsu and Takayanagi 2012). Once the process starts, a progressive recruitment of inflammatory T cells and macrophages into the joints occurs through a complex series of adhesion and migratory events. In this sense, initially there is an extensive increase in the number of innate effector cells in the synovial lining layer, which becomes several layers thick. Sublining layer becomes infiltrated with inflammatory mononuclear cells, including lymphocytes and macrophages and mast cells. In fact, macrophages are central effectors of synovitis and act though release of cytokines, reactive oxygen and nitrogen intermediates, Introducción 5 production of prostanoids and matrix-degrading enzymes, phagocytosis and antigen presentation. In addition, neutrophils transmigrate rapidly from the intravascular compartment to synovial fluid in the joint cavity contributing to the joint swelling and synthesizing prostaglandins, proteases and reactive oxygen intermediates (Cascao, Rosario et al. 2010). All of these cells, including resident fibroblasts, produce cytokines, which together with locally produced autoantibodies and immune complexes and complement, maintain the chronic inflammation leading the membrane expansion, which forms pannus. The cell layer of fibroblast- and macrophage-like cells which compose the pannus, can produce proteinases, receptor activator of nuclear factor kappa B ligand (RANKL) and other factors causing cartilage destruction. The persistent synthesis of proinflammatory cytokines plays a remarkable role in the development of RA disease from early autoimmunity step through continuous chronic inflammation into destruction of joint tissue. In addition, the prolonged immune activation is the main responsible to erosive and systemic bone loss and an increased propensity to fall in RA patients. The development of the inflammatory process in RA involves many different type of cells and a complex cytokine network. Upon activation and expansion, CD4 + T cells develop into different T helper cell subsets with different cytokine profile and distinct effector functions. Activated T cells that secret IFNϒ, IL2, IL12, IL18, TNF are produced in the synovial fluid and expressed in the synovial membrane. Particularly, TNF and IL-1β are considered as the main proinflammatory cytokines in the pathogenesis of RA. TNF plays a fundamental role through activation of cytokine and chemokine expression, expression of endothelial-cell adhesion molecules, protection of synovial fibroblasts, promotion of angiogenesis, suppression of regulatory T cells and induction of pain (McInnes and Schett 2011). In fact, treatment with TNF- α inhibitors results in decreased inflammation and bone protection in RA patients (Scott and Kingsley 2006). IL-1 family cytokines are also abundantly expressed in RA and promote the activation of leucocytes, endothelial cells, chondrocytes and osteoclasts (Brennan and McInnes Introducción 6 2008). Th17 cells, via their production of IL-17, contribute crucially to joint inflammation and bone erosion in RA (Kotake, Udagawa et al. 1999, Lubberts 2010, Jung, Kim et al. 2014). IL-6 and TGF-β induce Th17 development and IL-23 promotes Th17 cell expansion. In addition, these cytokines activate macrophages to secret other proinflammatory cytokines such as TNF and IL- 1β, IL-6, IL-12, RANKL expression on T cells, promote the differentiation of B cells and stimulate the release of matrix metalloproteases (MMP) provoking the degradation of the cartilage and the activation of osteoclasts leading to the bone resorption (Schett and Teitelbaum 2009, Chimenti, Triggianese et al. 2015). Figure 1. Adaptive and Innate Immune Processes within the Joint in Rheumatoid Arthritis (McInnes and Schett 2011) Understanding the intracellular targets that regulate cytokines in RA can potentially lead to new therapeutic interventions. In this sense, the inhibition of key signal transduction pathways in inflammation such as mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol-3 protein kinase (PI3) pathway, janus kinase-signal transducer and activator of transcription (Jak-STAT) and nuclear factor kappa B (NFKB) would be expected to Introducción 7 abolish the cell activation by cytokines and production of these proinflammatory cytokines in RA (Morel and Berenbaum 2004). For example, NF-κB is activated in the synovium of patients with RA and regulates genes that contribute to inflammation including TNF, IL-6, IL8, iNOS and COX-2 (Aupperle, Bennett et al. 2001). Similarly, MAPK have attracted considerable attention as potent therapeutic targets in RA. MAPK are also key regulators of cytokines and MMP production and could also targeted in RA. All three kinases families, c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), are expressed in rheumatoid synovial tissue (Schett, Tohidast-Akrad et al. 2000). 1.1. Pharmacological treatment of RA The current pharmacological therapies for the treatment of RA are designed to fast control the disease activity and prevent the progression of damage in tissues. These include early and strict control of the activity, the use of drugs for the treatment of moderate disease, and induction of remission with use of biological drugs. First line therapy including medications that suppress inflammation such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids; they act rapidly to improve pain and swelling due to RA. Disease-modifying antirheumatic drugs (DMARDs) are slower-acting compounds, not only improve symptoms but also diminish clinical and radiographic progression. In 1999, first biological-response modifier was marketed. These agents were designed to target the inflammatory mediators of tissue damage in RA (TNF-α, IL-1). The newest pharmacological target are anti-IL-6-receptor monoclonal antibodies. NSAIDs NSAIDs relieve the bone and joint inflammatory symptoms of RA; can be useful in the first weeks after the onset of RA symptoms while a diagnostic workup is undertaken, before a diagnosis is certain, and as bridge therapy while waiting for a slow-acting DMARD to become effective. The mechanism of action of NSAIDs is the inhibition of COX. COX is critical in the metabolism of arachidonic acid to the pro-inflammatory prostaglandins. Two isoforms of COX Introducción 8 have been extensively studied in humans: COX-1 and COX-2. COX-1 is expressed constitutively in the stomach, intestines, kidneys, and platelets and is involved in functions such as gastric protection from hydrochloric acid. COX-2, a predominantly inducible isoenzyme, is involved in the production of prostaglandins E2 and I2, which are upregulated in the inflammatory response (Vane and Botting 1998). NSAIDs are usually well tolerated for short periods of time; however, with chronic use they sometimes lead to gastrointestinal complications, such us ulcer formation, perforations, and bleeding (Chiba, Sato et al. 2005). Another potential adverse effect of NSAIDs is their renal toxicity. Glucocorticoids. Glucocorticoids have been widely used in the treatment of RA. Although high doses of glucocorticoids clearly cause unacceptable toxicity, daily doses of prednisone (or its equivalent) of ≤15 mg may diminish pain and swelling in many patients. This dosage of glucocorticoids may also have a limited disease-modifying effect in patients with RA (van Everdingen, Jacobs et al. 2002). First, they promote the expression of lipocortin-1, which inhibits the enzyme phospholipase A2 and the generation of arachidonic acid. Second, their transcription products inhibit the action of NF-κΒ and AP-1 protein, which act to upregulate proinflammatory molecules, like TNF and IL-1. Third, they exert an effect through membrane-associated receptors and second messengers-a nongenomic effect (De Bosscher, Schmitz et al. 1997). Glucocorticoids present a large number of adverse effects such us, diabetes, hypertension, peptic ulcer, among others. Given the slow onset of action of traditional DMARDs, low doses of glucocorticoids are often used as a bridge therapy to control symptoms until the DMARDs or biological agents become effective. Monotherapy with glucocorticoids is not recommended. DMARDs Methotrexate Methotrexate low dose is used as an immunosuppressant in autoimmune and rheumatic diseases. Methotrexate treatment has demonstrated effectiveness, reliability, sustained long- Introducción 9 term action and high tolerability. Up to four mechanisms of action have been proposed for the drug. First, it works as an antifolate agent (Quemeneur, Gerland et al. 2003). Second, methotrexate may diminish the accumulation of toxic compounds, especially polyamines, which contribute to tissue injury in RA (Cronstein 2005). Third, methotrexate may reduce intracellular levels of glutathione (Phillips, 2003). Finally, it increases extracellular adenosine levels, an antiinflammatory action (Montesinos, Desai et al. 2003). All of these mechanisms induce inhibition of the immune cellular proliferation and strong anti-inflammatory activity. Potential adverse effects of methotrexate include hepatitis and cirrhosis, oral ulcers, cytopenias, and interstitial pneumonitis. According these side effects, it is important monitoring patients receiving methotrexate treatment (Kremer, Alarcon et al. 1994). Hydroxychloroquine Hydroxychloroquine is an antimalarial that has been introduced as therapy for RA. The efficacy of hydroxychloroquine depends on the interference with antigen presentation; lysosomal membrane stabilization, and inhibition the metabolism of deoxyribonucleotides (Fox and Kang 1993, Weber and Levitz 2000). Hydroxychloroquine is rarely used as monotherapy for RA; its main clinical utility is in combination with other DMARDs. Sulfasalazine Sulfasalazine is cleaved in the intestine by bacterial organisms into 5-aminosalicylic acid and sulfapyridine. Sulfapyridine seems to exert the therapeutic action after intestinal absorption. The mechanism of action of sulfasalazine is based on neutrophil function inhibition, immunoglobulin levels reduction, and interference with T-cell function via suppression of NF-κB activation (Carlin, Djursater et al. 1992, Gadangi, Longaker et al. 1996). The most common toxicities are gastro intestinal-related (nausea, vomiting, diarrhea, abdominal pain) and hematologic in nature (neutropenia, thrombocytopenia), but the drug is generally considered safe and well-tolerated (Symmons, Salmon et al. 1988). Leflunomide Introducción 10 Leflunomide is an immunomodulatory drug, is a competitive inhibitor of dihydroorotate dehydrogenase, enzyme required for the de novo synthesis of pyrimidines, necessaries for the activation of lymphocytes. Therefore, blockade of the pyrimidine-synthesis pathway has antiproliferative effects. Leflunomide is generally well tolerated; hepatotoxicity and gastrointestinal intolerance are its most important adverse reaction (Olsen and Stein 2004). It is most commonly used as an alternative for methotrexate-intolerant patients. Other antirheumatic drugs Gold salts, their mechanism of action include the reduction of circulating B cells, immune complexes, rheumatoid factor, and immunoglobulin levels (Hirohata, Nakanishi et al. 1999). Long treatment with gold salts could present toxic reactions, including mucocutaneous reactions, proteinuria, and cytopenias. Minocycline, from tetracycline family, in addition to their antimicrobial action, it has antiinflammatory and immunomodulatory properties (O'Dell, Paulsen et al. 1999). Among its side effects, include autoimmune syndromes (serum sickness, polyarthritis, druginduced lupus, and vasculitis), headaches, GI intolerance, and a greying skin pigmentation. Cyclosporine, a calcineurin inhibitor demonstrated efficacy in the treatment of RA ( Sawitzke AD 2005). However, given the availability of safer DMARDs and biological agents, its role is now limited to a third-line agent after failure of first- and second-line drugs. Biological agents TNF- α antagonist TNF-α, an inflammatory cytokine, that is important in the pathogenesis of RA, binds to two receptors, the type 1 TNF receptor (p55) and the type 2 TNF receptor (p75), that are expressed on many types of cells. The biologic activity of TNF-α can be attenuated by soluble TNF receptors. Patients with RA have high concentrations of TNF-α in the synovial fluid. TNF-α is localized to the junction of the inflammatory pannus and healthy cartilage, and high synovial fluid TNF-α concentrations are associated with the erosion of bone (Olsen and Stein 2004). Introducción 11 Infliximab Infliximab is a chimeric molecule that joins the Fc region of human immunoglobulin G1 (IgG1) with the variable region of a mouse antibody against TNF-α (Elliot, Maini et al. 2008). It binds to soluble and membrane-bound TNF-α with high affinity, impairing the binding of TNF-α to its receptor; inducing a complement and antibody-dependent response against cells that express TNF-α. Infliximab can be used as monotherapy, but administration infliximab plus methotrexate is more effective (Lipsky, van der Heijde et al. 2000). Etanercept Etanercept is a soluble molecule composed of two recombinant p75 TNF-receptor proteins fused together to form a dimer (Nestorov 2005). Each of these molecules is linked to the Fc portion of human IgG1 to provide them a longer half-life. Etanercept is indicated in the treatment of refractory RA to others DMARDs included methotrexate. Adalimumab Adalimumab is a recombinant human IgG1 monoclonal antibody that binds to human TNF-α with high affinity, both impairing cytokine binding to its receptors and lysing cells that express TNF-a on their surface. It has a long circulating half-life (approximately 10–20 hours) and can be self-administered. Adalimumab is indicated in the treatment of refractory RA to others DMARDs included methotrexate. IL-1 antagonist Anakinra IL-1 is a pro-inflammatory cytokine produced by monocytes, macrophages, and some specialized cells in the synovial lining, has inflammatory effects that include the induction of IL- 6 and COX-2. In patients with rheumatoid arthritis, levels of IL-1-receptor antagonist in the damaged joint are diminished. Anakinra is a recombinant form of human interleukin- 1–receptor antagonist that targets the type I interleukin-1 receptor that is expressed in many tissues. Introducción 12 Anakinra in combination with methotrexate are indicated in patients who have not responded to methotrexate monotherapy (Cohen, Hurd et al. 2002). Anti-CD20 Rituximab Rituximab is a monoclonal antibody directed against an antigen, CD20, on the surface of all B cells other than stem cells and pre-B lymphocytes. It does not affect plasma cells since they do not possess CD20 on their cell surface. It exerts its effect by binding to CD20 on B cells and causing cell lysis by both complement-dependent and antibody-dependent cell mediated cytotoxicity. It has efficacy in RA patients who have failed conventional DMARDs and in RA patients who have failed anti- TNF agents (Edwards, Szczepanski et al. 2004, Cohen, Emery et al. 2006). Anti-CD80/86 Abatacept Abatacept is a fusion protein linking the extracellular domain of human cytotoxic T- lymphocyte associated antigen 4 (CTLA-4) to the Fc portion of human IgG1. It works by competing for the binding between CD28 on the T cell and CD80/86 on the antigen-presenting cell. This is an important co-stimulatory signal that is essential for T cell activation (Fan and Leong 2007). It is effective in patients who have an inadequate response to either methotrexate or one or more TNF-inhibitors (Genovese, Becker et al. 2005). IL-6 antagonist Tocilizumab Tocilizumab is a humanized anti-human IL-6 receptor antibody that specifically inhibits the biological activity of IL-6 by competitively inhibiting the binding of IL-6 to the IL-6 receptor (Paul-Pletzer 2006). Tocilizumab monotherapy has been shown to be effective in the treatment of RA and may be an option for patients that have side effects with combination biologics and DMARDs (Mima and Nishimoto 2008). Introducción 19 3.1.1. Benzoic acid derived Yoon et al. have shown anti-inflammatory effects of gallic acid using fibroblast-like synoviocytes (RA-FLS) cells isolated from the synovial tissues of patients with RA. They demonstrated that gallic acid could induce apoptosis in RA-FSL through regulation of apoptosis related protein expressions such as caspase-3, Bcl-2, Bax, p53 and pAkt. As well, gallic acid was able to reduce pro-inflammatory cytokines (IL-1β, IL-6), chemokines (CCL-2/MCP-1, CCL-7/MCP- 3), COX-2, and MMP-9 gene expression (Yoon, Chung et al. 2013). On the other hand, treatment with protocatechuic acid significantly reduced the paw swelling in adjuvant-induced arthritis (AIA) in rats. Additionally, these treatment could decease lipid peroxides (LPO) expression and reactive free radicals nitric oxide (NO) levels and increase antioxidant enzyme expression, such as, superoxide dismutase (SOD), catalase and glutathione (GSH) in liver. The antiarthritic activity of protocatechuic acid was found to be equivalent to that of diclofenac sodium (Lende, Kshirsagar et al. 2011). 3.1.2. Cinnamic acid derived It is well know that T cells are involved in initialling disease process and, even more, emerging evidence suggests that these cells also perpetuate RA pathogenesis at the late stage by secreting various mediators, such as receptor activator of NK-kB ligand (RANKL), IL-17 and osteopontin, which influence osteoclasts and FLS (Cho, Yoon et al. 2004, Xu, Nie et al. 2005). Rosmarinic acid has shown has apoptotic activity toward T cells from RA patients and further verified target T cell subsets. CD3+CD25+ activated T-cell subsets from most of the RA patients displayed significantly higher apoptosis rates than did the PBMCs and total CD3+ T cells. Furthermore, activated and effector CD4+ T cells, including CD4+CD25+ and CD4+CD45RO+ T cells, had a tendency of being more susceptible to rosmarinic acid-induced apoptosis than that of resting and naïve T-cell subsets. Rosmarinic acid was able to induce the release of cytochrome c from mitochondria and the blockage of mitochondrial depolarization inhibited apoptosis (Hur, Suh et al. 2007). On the other hand, Hsu et al., have reported that Rosmarinic acid decreased Introducción 20 the number of multinucleated osteoclasts in a dose-dependent manner using osteoclastprecursor cell line RAW264.7 stained with tartrate-resistant acid phosphatase (TRAP). The effects of this phenolic acid on osteclatogenesis were further confirmed by using primary murine bone marrow-derived macrophages (BMM) stimulated with RANKL. Rosmarinic acid was able to significantly reduced the c-fos, nuclear factor of activated T cells c1(NFATc1), TRAP, OSCAR, DCSTAMP, and ß3 integrin mRNA expression through inhibition of phosphorylation of p38 and ERK MAPKs and blocking the IkB degradation and supressing the subsequent nuclear translocation of NF-kB p65 (Hsu, Cheng et al. 2011). Similar activity showed chlorogenic acid who inhibited RANKL- mediated osteoclast differentiation by down-regulation of phosphorylation of p38, JNK and ERK MAPKs and IkB degradation. Chlorogenic acid, also supressed the mRNA expression of NFATc1, TRAP and OSCAR in RANKL-treated BMMs (Kwak, Lee et al. 2013). According to the in vivo studies, rosmarinic acid has been verified that ameliorated CIA, as manifested by reduction of synovitis and depletion of COX-2 positive cells in affected joints (Youn, Lee et al. 2003). The immunomodulatory effect of chlorogenic acid, was described by Chauhan et al. in AIA in rats. In this model, chlorogenic acid was able to control the total (CD3) and differentiated (CD4 and CD8) T cells count and supressed CD80/86 co-stimulatory molecule. Also this phenolic acid had the ability of diminish Th1 cytokines (IL-2, IFN-γ and IL-12) and elevate Th2 cytokines (IL-4 and IL-10) in CD4 + cells (Chauhan, Satti et al. 2012). p-cumaric acid also showed immunomodulatory and anti-inflammatory in AIA in rats. The cinnamic acid derived improved the arthritic disease through reduced the macrophage phagocytic index, circulating immune complex in sera and synovial TNF-α expression (Pragasam, Venkatesan et al. 2013). 3.2. Flavonoids 3.2.1. Flavones Apigenin and luteolin are flavones abundantly present in common fruits and vegetables such as chamomile tea and celery among others food. It has been demonstrated that apigenin has potential as antiarthritic agent using in vitro model (Shin, Kim et al. 2009). This dietaryplant Introducción 21 flavonoid was able to inhibit the proliferation of RA-FSLs by inducing apoptosis through activation of the effectors caspase-3 and caspase-7. Apigenin treatment resulted in activation of ERK MAPK, and when the cells were treated with the ERK inhibitor, apigenin-induced apoptosis was reduced. On the other hand, the authors demonstrated that the induction of the apoptosis and the activation of ERK by apigenin, it was due to the apigenin-induced generation of ROS (Shin, Kim et al. 2009). Similar results showed Sun et al., apigenin treatment was able to induce apoptosis of RA-FSLs, which was coupled with increased caspase-3 expression and activity and decreased Bcl-2/Bax ratio. These results were likely associated with downregulation of PI3-K/Akt activity (Sun, Jiang et al. 2012). Hou et al., reported that luteolin inhibited the proliferation of collagen-induced arthritis (CIA) rat synovial fibroblasts, which was accompanied by a decrease in MMP-1 and MMP-3 secretion. Besides, cytokine production (IL-6, IL-8, IL-15 and TGF-β) were found to be lower in CIA rat synovial fibroblasts treated with luteolin. The authors also demonstrated that luteolin treatment caused a delay of cells in the G2/M phase and this polyphenol inhibited the MAPK/ERKs and PI3K-Akt pathways (Hou, Wu et al. 2009). Other in vitro study on SW982 cell line, demonstrated that luteolin treatment resulted in inhibition of IL- 1β-induced MMP-1, MMP-3, TNF-α and IL-6 production. Moreover, IL-1b-induced activator protein-1 (AP-1) and NF-κB activation were inhibited by luteolin (Choi and Lee 2010). Nobiletin is a citrus flavone present especially in tangerines. Nobiletin shown anti-inflammatory effects since it was able to downregulate the production of MMP-1, MMP-3 and MMP-9 and PGE 2 in IL- 1 stimulated rabbit synovial fibroblast, moreover this citrus polyphenol, inhibited the proliferation of these cells (Ishiwa, Sato et al. 2000). This citrus flavone ameliorated CIA in mice. The reduced arthritis index and severity were accompanied by a suppression of osteoclastogenesis and TRAP activity in RAKNL-stimulated RAW264.7 cells. Moreover, nobiletin treatment resulted in inhibition of activation of MAPKs as well as AP-1/NF-kB pathways (Murakami, Song et al. 2007). On the other hand, in another CIA study, nobiletin was able to Introducción 22 improve the severity of damage and the authors, also demonstrated that this flavone reduced aggrecanase-1/a disintegrin and MMP with thrombospondin-like motifs (ADAMTS)-4 and ADAMTS-5 mRNA expression in CIA mice (Imada, Lin et al. 2008). 3.2.2. Flavonols Quercetin, kaempferol and myricetin are found in several foods, including onions, berries, grapes and red wine and exhibit a wide range of antiarhritic effects. Sato et al. demonstrated in 1997 that quercetin supresses IL-8 and MPC-1 mRNA in human synovial fibroblats stimulate with TNF-α. H 2 O 2 mediated induction of IL-8 and MCP-1 genes was also inhibited by quercetin in synovial fibroblats. Besides quercetin inhibited the induction of NF-kB by TNF-α (Sato, Miyazaki et al. 1997). Other study in RA-FLS treated with quercetin reported that quercetin inhibited the IL-1β-induced proliferation. In addition, quercetin downregulated the expression of MMP-1, MMP-3, COX-2 and production of PGE 2 in RA-SFs via inhibition of IL-1β- induced activation of NF-kB and phosphorylation of p38, JNK and ERK MAPKs pathway (Sung, Lee et al. 2012). The apoptotic pathway of quercetin in RA-SFs was described by Xiao et al., DNA fragmentation assay showed that quercetin elevated the apoptosis of RA-SFs, accompanying with enhanced caspase-3 and caspase-9 cleavages. Moreover, quercetin caused loss of mitochondrial membrane potential and cytochrome C release to cytosol and also decreased Bcl- 2/Bax ratio. Additionally, quercetin elevated phosphorylation at ser15. Indicating that quercetininduced apoptosis of RA-SFs was through mitochondrial pathway, in which p53 played an important role (Xiao, Hao et al. 2013). In vitro antioxidants studies have revealed the scavenging activity of ABTS and DPPH radicals, inhibition of nitric oxide and the superoxide radical scavenging capacity of quercetin (using thioglycolic acid-capped cadmium telluride quantum dots (QDs) as nanocarrier) (Jeyadevi, Sivasudha et al. 2013). Administration of QDs-quercetin complex showed a reduction in inflammation and improvement in cartilage regeneration in AIA in rats. Histology of hind limb tissue confirmed the complete cartilage regeneration in AIA rats treated with QDs-quercetin complex. This treatment reduced the expressions lipid peroxidation Introducción 23 and showed increase in activities of antioxidant enzymes such as SOD, GSH, glutathione peroxidase (GPx), catalase (CAT) levels in paw tissue. C-reactive protein, rheumatoid factor, red blood cells and white blood cells count and erythrocyte sedimentation rate of experimental animals were also brought back to normal levels (Jeyadevi, Sivasudha et al. 2013). Oral administration of quercetin in rat model of AIA ameliorated all markers of inflammation such as MCP-1, IL-1β and C-reactive protein and markers of oxidative stress such as 13/15 LOX restoring plasma antioxidant capacity. A protective effect of quercetin was verified being that HO-1 protein levels were upregulated in the animals treated. In this study, the authors deepened signalling pathways possibly involved, in this way, they demonstrated that quercetin was able to inhibit phosphorylation of ERK MAPKs and activation of NF-kB pathway (Gardi, Bauerova et al. 2015). On the other hand, kaempferol has also shown exert anti-inflammatory and antiarthritic proprieties. As describe Yoon et al., kaempferol inhibits IL-1β- induced proliferation of RA-SFs by apoptotic way. Likewise, kaempferol treatment downregulated the expression of MMP-1, MMP-3, COX-2 and the production of PGE 2 in IL-1β-stimulated RA-SFs. According to the involved pathways, this flavonol inhibited the phosphorylation of p38, JNK and ERK MAPKs and the activation of NF-kB signalling pathways (Yoon, Lee et al. 2013). A recent study testing a possible immunomodulatory activity of kaempferol in arthritis showed that kaempferol could strengthen the suppression function of Treg cells and prevent the pathological symptom of CIA in a rat model. Mechanistically, kaempferol treatment could stabilize FOXP3 protein level in vivo and in vitro. Furthermore, this polyphenol could inhibit PIM1-mediated FOXP3 phosphorylation at S422 to promote FOXP3 transcriptional activity (Lin, Luo et al. 2015). Finally, Lee et al., demonstrated that myricetin was able to decreased IL-1β- induced production of IL-6 and MMP-1 in SW982 syovial cells. Moreover, myricetin diminished the phosphorylation of p38 and JNK MAPKs (Lee and Choi 2010). Introducción 24 3.2.3. Flavanones Flavanones are polyphenolic compounds highly and almost exclusively present in citrus. Experimental observation have shown that these compounds, including hesperidin and naringin present anti-inflammatory and antiarthritic proprieties. Orally administration of hesperidin to CIA mice, result in improvement of clinical scores. Furthermore, hesperidin treatment was able to improve histological features such as reduced the damage of interchondral joints and supressed the increases of infiltration of inflammatory cells and pannus formation. This citrus flavonoid also, downregulated TNF-α mRNA expression in lesions (Kawaguchi, Maruyama et al. 2006). Similarly results, have been demonstrated by Li et al., using a model of AIA in rats. Hesperidin supressed secondary paw swelling and reduced the polyarthritis index of AIA rats. These macroscopic results were companied by amelioration of joint destruction and inhibition of bone destruction, synovial hyperplasia and inflammatory cells infiltration. In addition, hesperidin enhanced concanavalin-A (ConA)-induced T-lymphocyte proliferation and recovered ConA-induced IL-2 production by solenocytes in comparison with AIA control group. Production of inflammatory cytokines such as IL-1, IL-6 and TNF-α were also determinate in peritoneal macrophages from treated rats and stimulated with LPS. In this way, hesperidin was able to inhibit the production of these pro-inflammatory cytokines (Li, Li et al. 2008). Other study of hesperidin in CIA model in rats reveals the anti-inflammatory and antioxidant effects of this molecule. Hesperidin treatment supressed the evolution of CIA rats and ameliorated the changes at histological level and was able to restore damage alteration to a great extent. Administration of hesperidin showed a significant reduction in neutrophil activation and infiltration in the joints, also decreased TBARS concentration by inhibiting lipid peroxidation in the cartilage tissue. The antioxidant enzymes GSH and SOD were upregulated by hesperidin, and cartilage catalase activity and nitrite content were lowered (Umar, Kumar et al. 2013). Finally, the anti-inflammatory and antiarthritic effect of naringin were shown using an AIA rat model. In this study, administration of naringin supressed the paw swelling in AIA rats Introducción 25 and decreased histopathological changes of joint tissues. Moreover, naringin suppressed the production of TNF-α, IL-1β and IL-6 in serum of arthritic rats. TUNEL assay demonstrated that naringin induced apoptosis of AIA synovial cells via regulation of the protein expression of Bcl-2 and Bax (Zhu, Wang et al. 2015). 3.2.4. Isoflavones Soy bean isoflavones have been identified as phytochemical therapeutic flavonoids for the treatment of RA. Genistein and daidzein possess structural similarities with selective estrogen receptor modulators such as tamoxifen and synthetic isoflavones (Mohammad-Shahi, Haidari et al. 2011). In vitro studies have revealed the anti-inflammatory and antiarthritic proprieties of genistein in human synovial fibroblasts. Genistein inhibited anchorage-dependent proliferation of synovial cells induced by IL-1β, TNF-α, or EGF, virtually inhibited S-phase entry, caused cell cycle arrest in G1 restriction point and inhibited colony growth of rheumatoid synovial cells induced by EGF. Genistein also downregulated MMP-2 and MMP-9 expression induced by IL-1β or TNF-α (Zhang, Dong et al. 2012). Genistein treatment decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells. Furthermore, genistein prevented TNF-α-induced NF-κB translocation as well as phosphorylation of IκB kinase-α/β and IκBα, and also suppressed TNF-α-induced AMPK inhibition (Li, Li et al. 2014). In vivo studies have also shown antiarthritic effect of isoflavones. One study reported that genistein orally treatment in CIA rats, decreased primary paw inflammation. Dietary genistein exhibited a different pattern of immunomodulation, augmenting lymphocyte activation with increased IL-4 secretion, but decreased IFN-γ by cultured spleen lymphocytes subjected to CIA inflammation (Wang, Zhang et al. 2008). Treatment with genistein and daidzein in CIA rats resulted in not only a reduction in disease symptoms but also a delay in the onset of symptoms. The ear thickness in treated rats was lowered. Prevention of the tissue damage and joint inflammation was also observed following treatment with two soy isoflavones. Genistein and daidzein were able to reduce TNF-α, IL-6, adiponectin and leptin serum concentrations Introducción 26 (Mohammad-Shahi, Haidari et al. 2011). Mohammad-Shahi demonstrated that genistein and daidzein also played an important role on the extra-articular complications in CIA model in rats. In this model, both isoflavones were able to restore paraoxonase and arylesterase enzyme activity, which was decreased in serum from CIA rats. Paraoxonase low concentration is associated with a lower HDL- cholesterol and a higher level of malondialdehyde and C-reactive protein. Genistein and daidzein could also reverse the increased levels of malondialdehyde resulting from CIA (Mohammadshahi, Haidari et al. 2013). 3.2.5. Antocyanins Anthocyanin belongs to a subgroup of bioactive flavonoids responsible for the blue, purple, and red colour of many fruits, flowers, and vegetables such as grapes, pommegranate and berries. It has been shown to have protective effects in in vitro and in vivo arthritis models. Malvidin, major grape anthocyanin, was able to improve AIA clinical score; both therapeutic and preventive oral protocols of malvidin were efficient in reducing arthritic signs. Finally, the level of nitrites was decreased following both therapeutic and preventive treatment with malvidin from peritoneal macrophages. Thus confirming the reduction of macrophages inflammatory state in vivo in the presence of malvidin (Decendit, Mamani-Matsuda et al. 2013). Delphinidin, presents in cranberries and Concord grapes, was found to be a specific inhibitor of histone acetyltransferase. Delphinidin treatment inhibited NF-kB function by inhibiting acetylationdependent nuclear translocation of p65 in TNF-α-stimulated human rheumatoid arthritis synovial cell line (MH7A). Furthermore, this antocyanin efficiently suppressed TNF-α-induced expression of IL-6, COX-2, and IL-1β in MH7A cells (Seong, Yoo et al. 2011). Cyanidin-3-glucoside inhibited receptor activator of RANKL-mediated osteoclast and formation and downregulated the expression of osteoclast differentiation marker genes (Acp5, CtsK, Oscar, Tm7sf4, Atp6v0d2, and Nfatc1) Pretreatment differentiation with cyanidin-3-glucoside considerably reduced the induction of ERK, JNK, and p38 MAPKs by RANKL in osteoclast precursor cells. Furthermore, cyanidin-3-glucoside dramatically inhibited the expression of c-Fos and Nfatc1, which are Introducción 27 important transcription factors for osteoclast differentiation and activation. The formation of osteoclasts in coculture of bone marrow cells and calvaria-derived osteoblasts was also inhibited by cyanidin-3-glucoside treatment (Park, Gu et al. 2015). 3.2.6. Flavanols Tea polyphenols have received considerable public attention due to the positive association between tea consumption and beneficial health effects. The putative health benefits attributed to green tea (Camellia sinensis, Theaceace) is due to high concentrations of polyphenolic compounds known as catechins, including epigallocatechin-gallate (EGCG), epigallocatechin (EGC), epicatechin-gallate (ECG), and epicatechin (EC). One study showed that EGCG pretreatment inhibited both the constitutive and IL-1β- induced chemokine MCP-1/CCL2 production, regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production, growth-regulated oncogene (Gro-α/CXCL1) production, and epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5) production, and MMP-2 activation by RA-SFs (Ahmed, Pakozdi et al. 2006). This was achieved by EGCG via selective inhibition of the IL-1β-induced protein kinase Cδ and NF-κB pathways (Ahmed, Pakozdi et al. 2006). It was also shown, that EGCG was effective in inhibiting IL-1β-induced MMP-1, MMP-3, and MMP-13 in human tendon fibroblasts (Corps, Curry et al. 2004). Synovial fibroblast IL-6 production has been shown to inhibit bone formation and to concomitantly stimulate bone resorption and pannus formation (Cronstein 2007). Other study showed that EGCG pretreatment inhibits IL-1β-induced IL-6 and vascular endothelial growth factor synthesis in RA- SFs (Ahmed, Marotte et al. 2008). Yun et al., showed that EGCG treatment resulted in inhibition of TNFα-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA-SFs by inhibiting AP-1 DNA binding activity (Yun, Yoo et al. 2008). Ahmed et al., demonstrated that caspase-3 activation by EGCG suppressed RA-SFs growth, and this effect was mimicked by Akt and NF-κB inhibitors. Mcl-1 degradation by EGCG sensitized RA-SFs to TNFαinduced cleavage of poly ADP-ribose polymerase protein and apoptosis (Ahmed, Silverman et al. 2009). Introducción 28 EGCG-containing green tea extract in drinking water ameliorated CIA in mice. The reduced CIA incidence and severity was reflected in a marked inhibition of the inflammatory mediators COX- 2, IFN-γ, and TNF-α in arthritic joints of green tea-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were found to be lower in serum and arthritic joints of green tea-fed mice (Haqqi, Anthony et al. 1999). Morinobu et al., showed that EGCG treatment reduced bone resorption as determined by tartrate-resistant acid phosphatase positive multinucleated cells, bone resorption activity, and osteoblast-specific gene expression of the transcription factor NFATc1. They also studied the in vivo effect of EGCG in CIA mice, where administration of EGCG inhibited inflammation (Morinobu, Biao et al. 2008). Another study showed that EGCG ameliorated arthritis and macrophage infiltration, and caused a reduction in the amount of MCP-1/CCL2-synthesizing osteoblasts in rat CIA model (Lin, Chang et al. 2008). EGCG orally treatment ameliorated clinical symptoms, reduced histological scores in arthritic mice and also lowered serum type-II collagen IgG2a antibodies. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4+ Foxp3+ regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) expression by CD11b+ dendritic cells (DC). Joint homogenates from EGCG-fed mice exhibited increased levels of Nrf-2 and HO-1 compared with control CIA mice (Min, Yan et al. 2015). Intra-articular injection of EGCG in CIA mice, showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, by histomorphological scoring of the articular cartilage (Natarajan, Madhan et al. 2015). Administration of EGCG to rat adjuvant-induced arthritis resulted in an inhibition of IL-6 levels in the serum and joints of EGCG-treated animals. This study also showed that EGCG enhances the synthesis of soluble gp130 protein, an endogenous inhibitor of IL-6 signalling and trans-signalling. The inhibition of arthritis in EGCG-treated rats correlated to the reduction in MMP-2 activity in the joints compared with the activity level in arthritic rats (Ahmed, Marotte et al. 2008). Green tea extract administration in drinking water ameliorated rat adjuvant- Introducción 35 Tabla 1. In vitro studies of dietary polyphenols in RA. Polyphenol Source Experimental System Mechanism/s of Action Efficac ious [] Ref Phenolic acids Benzoic acid derived Gallic Acid Tea RA-FSL Gallic acid induces apoptosis of RA FLS through regulation of apoptosis related protein expressions (caspase-3, Bcl-2, Bax, p53 and pAkt) and reduces the expression of pro-inflammatory genes (IL-1β, IL- 6), chemokines (CCL-2/MCP-1, CCL-7/MCP-3), COX-2, and MMP-9. 0.1 and 1 µM ( Yoon, Chung et al. 2013) Cinnamic acid derived Rosmarinic Acid Basil, rosemary, thyme and peppermint RA PBMCs Murine bone marrow-derived macrophages Rosmarinic Acid induces the preferential apoptotic activity of activated T cells and effector T cells via mitochondrial pathway. Rosmarinic acid inhibits RANKL-mediated osteoclast differentiation by down-regulation MAPKs and NF-kB pathway and suppressing NFATc1, TRAP and OSCAR mRNA expression 50 µM 60 and 120 µM ( Hur, Suh et al. 2007) (Hsu, Cheng et al. 2011) Chlorogenic acid Peach, prunes and green cofee Murine bone marrow-derived macrophages Chlorogenic acid inhibits RANKL-mediated osteoclast differentiation by down-regulation MAPKs and NF-kB pathway and suppressing NFATc1, TRAP and OSCAR mRNA expression 28, 70 and 141 µM ( Kwak, Lee et al. 2013) Flavonoids Flavones Apigenin Parsley, celery and chamomile tea MH7A cells RA-FSL Apigenin induces apoptosis through activation of the effectors caspase-3 and caspase-7 and activation of ERK MAPK. Apigenin induces apoptosis of RA-FSL through increase caspase-3 expression and activity and decrease Bcl2/Bax ratio, mediated by downregulation of PI3-K/Akt activity 100 μM 20 µM ( Shin, Kim et al. 2009) (Sun, Jiang et al. 2012) Lu teolin Celery, broccoli, green pepper, parsley and thyme. Rat synovial fibroblasts SW982 Luteolin inhibits the proliferation of synovial fibroblasts in CIA rats. Decreases the secretion of MMP-1 and -3 and the expression of IL-6, IL-8, IL-15, and TGF-β, through inhibition of MAPK/ERKs and PI3K-Akt pathways. Treatment with luteolin significantly inhibited IL-1β- induced MMPs (MMP-1 and -3) and cytokines (TNF-α and IL-6) production. Luteolin also inhibited JNK and p38 MAPKs activation and the transcription factors AP-1 and NF-κB. 20 µM 1 and 10 µM ( Hou, Wu et al. 2009) (Choi and Lee 2010) Introducción 36 Nobiletin Tangerine Rabbit synovial fibroblasts and articular chondrocytes. RAW264.7 Nobiletin downregulates the production of MMP-1, MMP-3 and MMP-9 and PGE 2 and COX-2 of IL-- stimulated rabbit synovial cells and inhibits proliferation of rabbit synovial fibroblasts in the growth phase. Nobiletin suppresses RANKL-induced osteoclastogenesis in macrophages through inhibition of MAPKs and the resultant regulation of transcription factors (AP-1 and NFκB) 4, 8, 16, 32 µM 4, 20 and 50 µM ( Ishiwa, Sato et al. 2000) (Murakami, Song et al. 2007) Flavonols Quercetin Onion RA-FSL RA-FSL RA-FSL Quercetin suppresses TNF-α induced IL-8 and MCP-1 mRNA. H 2 O 2 mediated induction of IL-8 and MCP-1 genes are inhibited by quercetin. Quercetin inhibites the activation of NF-kappa B by TNF-α. Quercetin inhibits IL-1β- induced proliferation of RAFSL and downregulates MMP-1, MMP-3, COX-2 expression and PGE2 production. Through inhibition of ERK, p38, JNK phosphorylation and activation of NF-kB by IL-β Quercetin induces the apoptosis of RA-FSL, through enhances caspase-3 and caspase-9 cleavages and through mitochondrial pathway. 50, 100 and 200 µM 100 µM 100, 200 and 300 µM ( Sato, Miyazaki et al. 1997) (Sung, Lee et al. 2012) (Xiao, Hao et al. 2013) Kaempferol Apples, grapes, tomatoes, green tea, potatoes, onions, broccoli, Brussels sprouts, squash, cucumbers, lettuce, green beans, peaches, blackberries, raspberries, and spinach RA-FSL Kaempferol inhibites the proliferation of IL-1β- stimulated RA-FSL, as well as the mRNA and protein expression of MMP-1, MMP-3, COX-2 and PGE 2 induced by IL-1β. Through inhibition of ERK, p38, JNK phosphorylation and activation of NF-kB by IL-β. 100 µM ( Yoon, Lee et al. 2013) M yricetin Vegetables, fruits, nuts, berries, tea and red wine. SW982 Myricetin decreases IL-1β-induced production of IL-6 and MMP-1 in synovial cells. Moreover, myricetin diminishes the phosphorylation of JNK and p38 MAPKs. 10 µM ( Lee and Choi 2010) Isoflavones Genistein Lupin, fava beans, soybeans, kudzu, and psoralen RA-FSL MH7A cells Genistein inhibits proliferation of RA-FSL induced by IL-1β, TNF-α and EGF; also inhibits MMP-2 and MMP-9 expression induced by IL-1β or TNF-α . Genistein decreases the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells. Genistein prevented TNF-α-induced NF-κB translocation as well as phosphorylation of IκB kinase-α/β and IκBα, and suppressed TNF-α-induced AMPK inhibition. 37 µM 5, 10 and 20 µM ( Zhang, Dong et al. 2012) (Li, Li et al. 2014) Antocyanins Delphinidin Grapes MH7A cells Delphinidin inhibits TNF-α-induced NF-κB function and suppresses expression of IL-6, COX-2 and IL-1β. 10 and 30 µM ( Seong, Yoo et al. 2011) Cyanidin G rapes, bilberry, blackberry, Osteoclast precursor cells Cyanidin inhibits osteoclasts formation and downregulates expression of osteoclast 100 µM ( Park, Gu et al. 2015) Introducción 37 blueberry, cherry, cranberry differentiation marker genes, inhibiting phosphorylation of MAPKs Flavanols EGCG Green tea RA-FSL Human tendon fibroblast RA-FSL RA-FSL RA-FSL EGCG inhibits IL-1β-induced MCP-1/CCL2, RANTES/CCL5, Gro-α/CXCL1, ENA-78/CXCL5 production ans MMP-2 activation, via inhibition of protein kinase Cδ and NF-κB pathway. EGCG inhibits IL-1β-induced MMP-1, MMP-3 and MMP-13 EGCG inhibits IL-1β induced IL-6 and VEGF. EGCG inhibits TNF-α-induced MMP-1 and MMP-3 expression by inhibiting AP-1 DNA binding activity. EGCG inhibits TNF-α induced Mcl-1 protein expression. EGCG actives caspase 3 activity, mediated via down-regulation of the TNFα-induced Akt and NF-κB pathways 10 and 20 µM 2.5 and 25 µM 10 and 200 µM 0.12, 0.25 and 0.5 µM 50 µM ( Ahmed, Pakozdi et al. 2006) (Corps, Curry et al. 2004) (Ahmed, Marotte et al. 2008) (Yun, Yoo et al. 2008) (Ahmed, Silverman et al. 2009) Diarylheptanoids a nd arylalkanones Gingerol Ginger ( Zingiber officinale) RA-FSL Ginger extract inhibits TNF-α-induced IL-8 and IL-6 ( Ribel - Madsen, Bartels et al. 2012) Curcumin Curcuma longa Human chondrocytes Co-culture canine mesenchymal stem cells and primary chondrocytes RA-FSL RA-FSL, MH7A HIG-82 rabbit synoviocytes Curcumin relieves suppression of collagen type II, β1-integrin and caspase-3 activation. Curcumin enhances the production of collagen type II, CSPGs, β1-integrin and activating MAPKs signaling and suppressing caspase-3 and COX-2. Curcumin induces apoptosis in RA-FSL by inhibition of Blc-2 and X-IAP expression, activation of caspase-3 and caspase-9 and degradation of PARP. Also, inhibits COX-2 expression and PGE 2 production. Curcumin blocks IL-1β and PMA-induced IL-6 expression, blocks PMA-induced VEGF expression, blocks activation of NF-κB and ERK1/2 and induces cell death through apoptosis. Curcumin inhibits inflammatory processes through suppression of collagenase and stromelysin expression. 50 µM 5 µM 50, 75 and 100 µM 12.5, 25 and 50 µM 1 and 10 µM ( Shakibaei, Schulze- Tanzil et al. 2005) (Buhrmann, Mobasheri et al. 2010) (Park, Moon et al. 2007) (Kloesch, Becker et al. 2013) (Jackson, Higo et al. 2006) Stilbenes Resveratrol Grapes RA-FSL Resveratrol induces apoptosis through caspase-3, caspase-9, caspase-8 and PARP cleavage, 100 µM ( Byun, Song et al. 2008) Introducción 38 Table 2. In vivo studies of dietary polyphenols in RA. Polyphenol Source Experimenta l System Mechanism/s of Action Efficacious [] Ref Phenolic acids Bezoic acid derived Protocatechuic acid Onions, mushroom. AIA in rats Protocatechuic acid inhibits arthritis index, decreases LPO and NO and increases antioxidants, SOD, catalase and GSH in liver. 50 and 100 mg/Kg orally ( Lende, Kshirsagar et al. 2011) Cinnamic acid derived p - Coumaric Acid Peanuts, tomatoes, carrots and garlic. AIA in rats p-coumaric acid reduces the serum circulating immune complex levels. Synovial tissue of arthritic rat treated with p-coumaric acid showed a reduction in immunostaining for TNF-α. 100 mg/kg i.p. ( Pragasam, Venkatesan et al. 2013) Chlorogenic Acid Peach, prunes and green coffee AIA in rats Chlorogenic acid controls the total (CD3) and differentiated (CD4 and CD8) T cells count, suppresses CD80/86 and suppresses the Th1 cytokines but elevates Th2 cytokines. 2.5, 5, 10, 20 and 40mg/kg. orally ( Chauhan, Satti et al. 2012) Rosmaric acid Basil, rosemary, thyme and peppermint CIA in mice Rosmaric acid reduces the arthritic index, histological damage and COX-2 expression. 50 mg/Kg i.p. ( Youn, Lee et al. 2003) Flavonoids Flavones MH7A cells RA-FSL RA-FLS mitochondrial cytochrome c release and AIF translocation to the nucleus. Resveratrol induces apoptosis through activation of caspase-3 and caspase-9, disrupts mitochondrial membrane potentials as a result of decreased Bcl-XL expression, allowing cytochrome c from the mitochondria into the cytosol, in a sirtuin 1- dependent manner. Resveratrol attenuates IL-1β-induced production of MMP-1, MMP-3 and MMP-9. qRT-PCR showed a significant reduction in the relative abundance of the transcripts of OPG and RANKL. Resveratrol attenuates TNF-α-induced production of IL-1β and MMP-3 via inhibition of PI3K-Akt signaling pathway in RA FLS. 100 µM 6.25, 12.5, 25 and 50 µM 100 µM (Nakayama, Yaguchi et al. 2012) (Glehr, Breisach et al. 2013) (Tian, Chen et al. 2013) Other dietary polyphenols Oleocanthal EVOO ATDC-5 ATDC-5 Oleocanthal inhibits LPS-induced NO production and iNOS expression Oleocanthal suppresses LPS-induced IL-6 and MIP-1α expression. 10 and 25 µM 15 µM ( Iacono, Gomez et al. 2010) (Scotece, Gomez et al. 2012) Introducción 39 Nobiletin Tangerine CIA in mice CIA in mice Nobiletin suppresses arthritic index. Nobiletin improves arthritic progression and joint histology and exerts a chondroprotective action from the aggrecanase-mediated cartilage destruction according to the inhibition of gene expression and production of ADAMTS-4 and -5 2 and 20 mg/Kg i.p. 15, 30 and 60 mg/Kg i.p. ( Murakami, Song et al. 2007) (Imada, Lin et al. 2008) Flavonols Quercetin Onions AIA in rats AIA in rats Quercetin improves arthritic progression and joint histology, decreases lipid peroxidation and increases antioxidants, SOD, catalase, GSH and GPx. Quercetin reduces MCP-1, IL-1β, C-reactive protein and 13/15 LOX. Upregulates HO-1 expression and inhibits ERK phosphorylation and NF-κB activation. 0.2 and 0.4 mg/Kg into thioglycolica cidcappedcadm iumtelluride quantumdot s. Orally 150 mg/Kg Orally ( Jeyadevi, Sivasudha et al. 2013) (Gardi, Bauerova et al. 2015) K aempferol Apples, grapes, tomatoes, green tea, potatoes, onions, broccoli, Brussels sprouts, squash, cucumbers, lettuce, green beans, peaches, blackberries, raspberries, and spinach CIA in rats Kaempferol inhibits PIM1-mediated FOXP3 phosphorylation at S422 to promote FOXP3 transcriptional activity. 100 mg/Kg ( Lin, Luo et al. 2015) Flavanones Hesperidin Orange and lemon CIA in mice AIA in rats CIA in rats Hesperidin improves clinical socore, histological features and downregulates TNF-α mRNA expression. Hesperidin reduces polyarthritis index and histological features. Enhances CoA-induced T- lymphocyte proliferation and recovers IL-2 production in solenocytes; also, inhibits IL-1, IL-6 and TNF-α level in peritoneal macrophages from treated rats. Hesperidin improves clinical socore, histological features and decrases TBARS concentration by inhibition of lipid peroxidation in the cartilage. Upregulates GSH and SOD antioxidant enzymes and downregulates cartilage catalase activity and nitrite level. 150 mg/Kg Orally 40, 80 and 160 mg/Kg Intragastrica lly 160 mg/Kg Orally ( Kawaguchi , Maruyama et al. 2006) (Li, Li et al. 2008) (Umar, Kumar et al. 2013) Naringin Grapes AIA in rats Naringin suppresses paw swelling and histopathological changes of joint tissue. Downregulates TNF-α, IL-1β and IL-6 serum level 20 and 40 mg/Kg Orally ( Zhu, Wang et al. 2015) Introducción 40 and induces apoptosis via regulation of Bcl-2 and Bax protein expression. Isoflavones Genistein Lupin, fava beans, soybeans, kudzu, and psoralen CIA in rats CIA in rats CIA in rats Genistein modulates immune responses in CIA model. Suppresses the secretion of IFN-γ and augments the IL-4 production. Genistein improves disease symptoms and reduces TNF-α, IL-6, adiponectin and leptin serum concentration. Genistein restores paraoxonase and arylesterase activity and decreases MDA levels. 80 mg/Kg Orally 20 mg/Kg Orally 20 mg/Kg Orally ( Wang, Zhang et al. 2008) (Mohamma d-Shahi, Haidari et al. 2011) (Mohamma dshahi, Haidari et al. 2013) Dai d zein Soybeans CIA in rats CIA in rats Daidzein improves disease symptoms and reduces TNF-α, IL-6, adiponectin and leptin serum concentration. Daidzein restores paraoxonase and arylesterase activity and decreases MDA levels. 20 mg/Kg Orally 20 mg/Kg Orally ( Mo hamma d-Shahi, Haidari et al. 2011) (Mohamma dshahi, Haidari et al. 2013) Antocyanins Malvidin Red wine AIA in rats Malvidin is efficient in reducing arthritis sings and decreases nitrites level from peritoneal macrophages from treated rats. 25 mg/Kg Orally ( Decendit, Mamani- Matsuda et al. 2013) Flavanols EGCG Green tea CIA in mice CIA in mice CIA in rats CIA in mice CIA in mice EGCG-containing green tea extract reduces disease symptoms, inhibits COX-2, IFN-γ and TNF- α level in arthritic joint and decreases anti-CII IgG serum level. EGCG ameliorates arthritis during the disease course and reduces histologic scores of arthritis. Also reduces the number of osteoclasts in the pannus of arthritic paw joint. EGCG reduces the severity of CIA and alleviates joint destruction. Moreover, diminishes the number of CCL2-producing osteoblasts and recruitment of CD68+ macrophages. EGCG ameliorates clinical symptoms, histological scores in arthritis, IL-1β, IL-6, TNF-α, IFN-γ, IL-10 and anti-CII IgG serum level. Also supresses T cell proliferation and increases Nrf2 and HO-1 levels. EGCG reduces histopathological cartilage degradation. 2 g/L of green tea extract in water, ad libitum as the sole source of drinking water. 20 mg/Kg i.p. 20 mg/Kg i.p. 10 mg/Kg Orally 12 mg/Kg Intraarticular injection ( Haqqi, Anthony et al. 1999) (Morinobu, Biao et al. 2008) (Lin, Chang et al. 2008) (Min, Yan et al. 2015) Min 201 (Natarajan, Madhan et al. 2015) Introducción 41 AIA in rats AIA in rats EGCG inhibits IL-6 and MMP-2production. EGCG enhances the synthesis of gp130 protein and endogenous inhibitor of IL-6 signalling. Green tea extract ameliorates arthritic damage via inhibition of IL-17 and upregulation of IL-10 serum level. 100 mg/Kg i.p. 8 and 12 g/L green tea extract in drinking warter (Ahmed, Marotte et al. 2008) (Kim, Rajaiah et al. 2008) Diarylheptanoids and arylalkanones Ginger Ginger (Zingiber officinale) CIA in rats Ginger ameliorates the clinical score, cartilage destruction and decreases serum levels of IL-1β, TNF-α, IL-2, IL-6 and anti-CII IgG. 50, 100 and 200 mg/Kg i.p. ( Fo uda and Berika 2009) Curcumin Curcuma longa CIA in mice AIA in rats AIA in rats Curcumin attenuates arthritic index and reduces BAFF, IFN-γ and IL-6 sera levels. Curcumin ameliorates symptoms of arthritis and preserves radiological alterations. Curcumin reduces TNF-α, MDA, nitrites C-reactive protein and citrullinated peptide antibody sera levels. Curcumin improves histopathological changes, decreases IL-1β and TNF-α synovial and sera levels and inhibit NF-κB pathway. 50 mg/Kg i.p. 10 and 30 mg/Kg Curcumin loaded solid lipid nanoparticle s. Orally 50 mg/Kg i.v. or orally (nanoformul ation) ( Huang, Xu et al. 2013) (Arora, Kuhad et al. 2015) (Zheng, Sun et al. 2015) Stilbenes Resveratrol Grapes Intraarticular injection of LPS in rabbits CIA in mice AIA in rats Resveratrol decreases cartilage destruction and loss of matrix proteoglycan content in the cartilage. Resveratrol attenuates clinical parameters and bone erosion; associated with markedly reduced serum levels of pro-inflammatory cytokines and collagen-specific IgG and with reduced numbers of Th17 cells and the production of IL-17 in DLN. Resveratrol reduces cartilage degeneration, reduces the production of COX-2 and PGE 2 and inhibits the proliferation of Con-A spleen cells 2.3 mg/Kg Intraarticula r injection 20 mg/Kg i.p. 10 and 50 mg/Kg Intragastrica lly ( Elmali, Baysal et al. 2007) (Xuzhu, Komai- Koma et al. 2012) (Chen, Lu et al. 2014) Other dietary polyphenols Punicalagin Pomegranate AIA in rats Punicalagin diminishes disease development and paw edema 10 and 50 mg/Kg i.p. ( Jean - Gilles, Li et al. 2013) Ellagic acid Blackberries, cranberries, pecans, pomegranate s, raspberries, strawberries, walnuts, wolfberries, and grapes CFA-induced polyarthritis in rats Ellagic acid reduces joint inflammation and histopathological changes; also suppresses TNF-α and IL-1β production. 0.13, 0.325 and 0.65% Topically ( Mo, Panichayup akaranant et al. 2013) Introducción 42 Oleuropein EVOO CIA in mice Oleuropein ameliorates clinical symptoms and histological scores in arthritis; also decreases proinflammatory cytokines and chemokine plasma levels and MPo activity. 40 mg/Kg i.p. ( Impellizzer i, Esposito et al. 2011) Hydroxytyrosol EVOO CIA in rats Hydroxytyrosol decreases paw edema and histological damage and reduces COX-2 and iNOS expression. 5 mg/Kg Orally ( Silva, Sepodes et al. 2015) JUSTIFICACIÓN Y OBJETIVOS Justificación y Objetivos 45 Según la Organización Mundial de la Salud, las enfermedades reumáticas son la causa más frecuente de incapacidad, de origen no mental, en el mundo. En España estas patologías están relacionadas con el 50.7% de las incapacidades laborales y son la principal causa de bajas laborales permanentes. Especial relevancia tiene la artritis reumatoide (AR), por sus repercusiones sobre la calidad de vida de los pacientes y el deterioro progresivo que produce esta enfermedad. Entre el 20% y el 30% de los pacientes con AR se convierten en incapacitados permanentes. Demográficamente, la AR es la forma más común de artritis inflamatoria y afecta aproximadamente al0.5 - 1% de la población mundial, con un impacto económico comparable con el de la enfermedad arterial coronaria. Esta patología puede aparecer en cualquier etapa de la vida, sin embargo la prevalencia aumenta con la edad de tal forma que el 80% de todos los pacientes inician la enfermedad entre los 35 y 50 años de edad y es unas 3 veces mayor en mujeres que en hombres (Gonzalez Cernadas, Rodriguez-Romero et al. 2014). La AR es una enfermedad autoinmunológica inflamatoria, crónica y sistémica que se caracteriza por una sinovitis erosiva simétrica, en la cual el tejido de granulación de origen sinovial (pannus), invade y erosiona el cartílago y el hueso de las articulaciones diartrodiales. La enfermedad se desarrolla con cifras elevadas de la velocidad de sedimentación eritrocitaria, proteína C reactiva, presencia de inmunocomplejos y el factor reumatoide. Los pacientes con AR presentan afectación generalmente de las articulaciones diartrodiales de forma simétrica. Dicha enfermedad tiene, en ocasiones, un componente extraarticular, afectando a diversos órganos y sistemas. El deterioro progresivo, articular o extraarticular, acaba provocando que estos pacientes presenten deformidad, dolor, incapacidad funcional y disminución de la expectativa de vida (Fauci 2010). La etiología de AR se desconoce aunque factores genéticos, infecciosos (infección viral, e.g. Virus Epsein-Barr, Proteus, rubeola,) ambientales (tabaco, situaciones de estrés y traumatismo, obesidad, dieta) y hormonales parecen estar involucrados en vías relacionadas y complejas (Suzuki and Yamamoto 2015). Se han identificado múltiples genes que contribuyen a la predisposición y susceptibilidad a la enfermedad fundamentalmente el gen HLA-DRB1 del complejo mayor de histocompatibilidad, PADI4, PTPN22, CCR6 y FCRL3, entre otros (Yamamoto, Okada et al. 2015). Aunque la AR ha sido considerada como una patología autoinmune, no existe consenso sobre la naturaleza del autoantígeno(s) o factores ambientales que inicia la desregulación de la respuesta inmune, mayormente orquestados por macrófagos, células T reguladoras (Treg), T Capítulo I 53 LA DIETA DE ACEITE DE OLIVE VIRGEN EXTRA PREVIENE LA RESPUESTA INFLAMATORIA Y LA DEGRADACIÓN DE LA MATRIZ DEL CARTÍLAGO EN UN MODELO DE ARTRITIS INDUCIDA POR COLÁGENO TIPO II EN RATONES MªÁngeles Rosillo, Marina Sánchez-Hidalgo, Susana Sánchez-Fidalgo, Marina Aparicio-Soto, Isabel Villegas, Catalina Alarcón-de-la-Lastra Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, España RESUMEN Antecedentes del tema: Aunque la patogenia de la artritis reumatoide (AR) sigue sin conocerse en su totalidad, actualmente se ha descrito la existencia de una desregulación en el balance Th1/Th2 y Th17/células T reguladoras en pacientes con AR. En las articulaciones artríticas, se produce un incremento en la expresión de citocinas proinflamatorias, tales como TNF-α, IL-1β o IL-17, que juegan un papel destacado en el desarrollo y progresión de la enfermedad. Este aumento se atribuye, al menos en parte, a una desregulación en las rutas de señalización celular protagonizadas por las MAP cinasas (MAPKs), JAK/STAT y el factor nuclear NF-κB. Por otro lado, el factor nuclear Nrf2, es una pieza clave en la transcripción de distintas enzymas antioxidantes como la hemo oxigenasa 1 (HO-1), se ha demostrado que la activación de la expresión génica de esta proteína es capaz de modular la respuesta inflamatoria. El hecho de que el abordaje farmacológico de la AR implique severos efectos secundarios, ha generado en los últimos años un auge en el uso de otras estrategias terapéuticas, entre las que destaca la terapia nutricional. Recientes estudios epidemiológicos han confirmado que el consumo de aceite de oliva virgen extra (AOVE), dentro del contexto de la dieta Mediterránea, resulta eficaz en la prevención de ciertas patologías relacionadas con el estrés oxidativo, la inflamación y el sistema inmune. Por otro lado, recientes publicaciones de nuestro grupo de investigación, así como de otros autores, han demostrado las propiedades antiinflamatorias e inmunomoduladoras de la dieta de AOVE en distintos modelos experimentales de inflamación. Objetivos: En base a los anteriores antecedentes, nos planteamos evaluar la influencia de una dieta de AOVE de la variedad picual, en un modelo de artritis experimental inducida por colágeno tipo II en ratones. Material y Métodos: Ratones DBA-1/J recién destetados, fueron distribuidos en cuatro grupos experimentales: (1) Ratones sanos alimentados con dieta de aceite de girasol (CS-AG), (2) Ratones artríticos alimentados con dieta de aceite de girasol (CIA-AG), (3) Ratones sanos Capítulo I 54 alimentados con dieta de AOVE (CS-AOVE), (4) Ratones artríticos alimentados con dieta de AOVE. Después de seis semanas, la artritis fue inducida. El día 0, los ratones fueron inmunizados con una inyección en la base de la cola de 100 mg de colágeno II. El día 21, recibieron una inyección de refuerzo intraperitoneal, con la misma cantidad de colágeno. El grado de desarrollo de la enfermedad fue valorado de forma visual siguiendo una escala de 0-2, dónde 0=sin inflamación; 1=inflamación leve; 1.5=inflamación marcada; 2=inflamación severa y según el estudio histológico de las articulaciones. Los niveles en suero de la proteína oligomérica de la matriz del cartílago (COMP) y de metaloproteasa 3 (MMP-3) así como los niveles de citocinas proinflamatorias como TNF-α, IL-1β e IL-17 en el homogenado de pata fueron determinados mediante la técnica de ELISA. Los cambios en la expresion proteica de HO-1, Nrf2 y de las proteínas implicadas en las vías de señalización celular MAPKs, JAK/STAT y NF-κB fueron estudiadas mediante western blot. Resultados: la dieta de AOVE redujo de forma significativa el edema articular y la destrucción del cartílago, previniendo el desarrollo de la AR. Los niveles séricos de COMP y MMP-3, los encontramos disminuidos en aquellos animales alimentados con la dieta de AOVE. La expresión de las citocinas proinflamatorias TNF-α, IL-1β e IL-17 fue menor el tejido articular de los ratones artríticos que recibieron la dieta de AOVE, en comparación con los CIA-AG. Además la activación de las vías de señalización MAPKs, JAK/STAT y NF-κB fue inhibida por la dieta de AOVE. De acuerdo con la expresión proteica de Nrf2 y HO-1, estuvo aumentada en las articulaciones de los ratones pertenecientes al grupo CIA-AOVE. Conclusión: Estos resultados confirman, por primera vez, que el AOVE muestra un elevado efecto protector en la prevención y la progresión de la AR experimental, probablemente a través de la activación de la vía Nrf2/HO-1, e inactivación de las vías MAPKs, JAK/STAT y NF-κB, con los que podría ser considerado como un alimento funcional de primera magnitud dentro de la terapia nutricional de pacientes con AR, si futuros ensayos clínicos confirmasen estos resultados experimentales. 1 3 Eur J Nutr DOI 10.1007/s00394-015-0850-0 ORIGINAL CONTRIBUTION Dietary extra‑virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen‑induced arthritis María Angeles Rosillo · Marina Sánchez‑Hidalgo · Susana Sánchez‑Fidalgo · Marina Aparicio‑Soto · Isabel Villegas · Catalina Alarcón‑de‑la‑Lastra Received: 28 August 2014 / Accepted: 13 November 2014 © Springer-Verlag Berlin Heidelberg 2015 of JAK/STAT, MAPKs and NF-κB pathways was drastically ameliorated. According to Nrf2 and HO-1, the protein expressions were up-regulated in those mice fed with EVOO. Conclusion These results support the interest of EVOO as a beneficial functional food to prevent the development of the rheumatoid arthritis (RA). Keywords CIA · EVOO · Inflammatory response · Olive oil · Rheumatoid arthritis Introduction Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial membranes and proliferation of the synovial lining leading to synovial hyperplasia and massive infiltration of immune cells, vasculogenesis, cartilage and bone destruction and progressive joint damage [1]. Although the pathogenesis of RA is not fully understood, genetic and immunologic factors clearly contribute to the joint damage during RA [2]. In fact, dysregulated balances of Th1/Th2 and Th17/Treg cells have been demonstrated in RA patients [3]. RA synovium is characterized by up-regulated proinflammatory cytokines levels such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β, the two key cytokines involved in destructive arthritis in addition to IL-17, IL-21, IL-23 and IL-6, among others. In fact, IL-17 plays important roles in the additive/synergistic effects induced together with TNF-α and IL-1β, and cartilage matrix breakdown induction through the chondrocyte metabolism dysregulation. Besides, cytokines contribute to the osteoclasts and macrophages activation and also promotion of recruitment of both neutrophils and monocytes Abstract Purpose Current experimental studies support a beneficial role of extra-virgin olive oil (EVOO) in several inflammatory diseases. The present study was designed to evaluate the effects of dietary EVOO on type II collagen-induced arthritis (CIA) in mice. Methods DBA-1/J mice were randomized in four experimental groups (10 or 15 animals per group): (1) Sham sunflower diet (SO-Sham), (2) CIA sunflower diet (SO-CIA), (3) Sham EVOO diet (EVOO-Sham) and (4) CIA EVOO diet (EVOO–CIA) group. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrified 42 days after first immunization. In addition to macroscopic and histological analyses, serum levels of cartilage olimeric matrix protein (COMP), metalloproteinase-3 (MMP-3) and pro-inflammatory cytokines levels were evaluated by ELISA. The expressions of heme oxygenase-1 (HO-1), nuclear factor E2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), Janus kinase-signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. Results EVOO diet significantly reduced joint edema and cartilage destruction, preventing the arthritis development. Dietary EVOO significantly decreased serum COMP and MMP-3 levels, as well as, the pro-inflammatory cytokines levels (TNF-α, IL-1β and IL-17). Moreover, the activation M. A. Rosillo (*) · M. Sánchez-Hidalgo · S. Sánchez-Fidalgo · M. Aparicio-Soto · I. Villegas · C. Alarcón-de-la-Lastra Department of Pharmacology, Faculty of Pharmacy, University of Seville, Profesor García González, 2, 41012 Seville, Spain e-mail: [email protected] C. Alarcón-de-la-Lastra e-mail: [email protected] Eur J Nutr 1 3 by means of inducing various chemokines. The infiltrated leukocytes produce more cytokines and tissue-destructive enzymes leading to chronic inflammation, cartilage damage and bone erosion [4]. The process of gene expression of these pro-inflam- matory mediators involves multiple signal transduction pathways including mitogen-activated protein kinases (MAPKs) [5, 6] and nuclear transcription factor-kappa B (NF-κB) pathways [6, 7]. Actually, NF-κB activation results in metalloproteinases (MMPs) production, which degrade articular extracellular matrix constituents and subsequently cause articular destruction. In addition, the Janus kinase–signal transducer and activator of transcription (JAK-STAT) is another relevant inflammatory pathway activated in response to cytokines [8]. In particular, STAT-3 overexpression has been reported in synovial membranes from RA patients [9]. Likewise, nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor orchestrator of the induction of several antioxidant enzymes, such as heme oxygenase (HO)-1. The activation of HO-1 gene expression is considered to be an adaptive cellular response to survive exposure to environmental stresses. More recently, HO-1 has also been reported to modulate inflammatory responses, cell proliferation and prevent apoptosis [10, 11]. Recent research has suggested that dietary patterns, such as the traditional Mediterranean diet, which is characterized by large amounts of foods naturally derived, in particular, vegetables, fruits, nuts, fish and grains, may confer protection from certain chronic diseases related to oxidative stress, inflammation and the immune system. In comparison with other healthy diets, Mediterranean diet has a high content of total fat as its most distinctive feature due to the usual high intake of olive oil from olive tree, Olea europaea [12]. Extra-virgin olive oil (EVOO) is obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to oil alteration. Traditionally, the beneficial effects of EVOO have been attributed to its high MUFA content (oleic acid) as it protects lipoproteins and cellular membranes, from oxidative damage [13]; however, nowadays, there is increasing evidence to support the beneficial effects of EVOO intake that are due to its minor highly bioactive components (about 1–2 % of oil weight) including phenolic compounds, triterpene alcohols, sterols, hydrocarbons (squalene), vitamins (α- and γ-tocopherols), β-carotene, phytosterols, among others [12, 14]. Particularly, current experimental studies support a beneficial role of polyphenols from EVOO in several inflammatory diseases, including RA [15–18]. In fact, our research group has previously demonstrated that an EVOO diet enriched with hydroxytyrosol, a phenolic compound present in the oil, and a dietary EVOO-polyphenol extract supplementation could improve the inflammatory responses associated with a chronic experimental colitis model [19, 20]. Furthermore, we also confirmed beneficial effects of dietary EVOO-unsaponifiable supplementation on an acute experimental colitis model [21]. Although dietary EVOO has demonstrated immunomodulatory and antiinflammatory properties in several inflammation experimental models [22–24], nevertheless its possible contribution to RA remains unknown. Taken this background into account, the present study was designed to evaluate the potential protective effects of dietary EVOO in collagen-induced arthritis (CIA) model, a well-studied animal model of RA useful in the development of new therapies for RA [10], and to identify its underlying molecular mechanisms. The severity of arthritis was assessed by macroscopic and histology parameters. Inflammatory mediators and the signaling pathways involved were also explored. Materials and methods Animals and diets A total of 50 three-week-old male DBA-1/J mice (Janvier®, Le Genest St Isle, France) were maintained in our Animal Laboratory Center under standard conditions (temperature, 24–25 °C; humidity, 70–75 %; lighting regimen, 12L/12D). They were fed pellet diets and water ad libitum. Mice were randomized into four experimental groups (10 or 15 animals per group) during all experimental period: (1) Sham sunflower diet (SO-Sham) group received a diet elaborated with a marketable sunflower oil (Koipesol®, DEOLEO, Spain), (2) CIA sunflower diet (SO-CIA) group received a diet elaborated with a marketable sunflower oil (Koipesol®, DEOLEO, Spain), (3) Sham EVOO diet (EVOO- Sham) group were fed with a diet made with a marketable EVOO picual variety characterized by a high content of polyphenolic compounds (600 ppm) (Oleoestepa®, Seville, Spain) and (4) CIA EVOO diet (EVOO-CIA) group were fed with a diet made with a marketable EVOO picual variety, containing high levels of polyphenolic compounds (600 ppm) (Oleoestepa®, Seville, Spain). All diets were formulated on the basis of the American Institute of Nutrition (AIN) standard reference diet with the modification of various sources of carbohydrate (Table 1), at 10 % of total oil (Table 2) and were prepared by mixing the respective compounds under yellow light and stored at −80 °C. Fresh diets were provided daily. The animals were fed with the corresponding diet during 6 weeks previous to the CIA induction and during the experiment time. Experiments followed a protocol approved by the Animal Ethics Committee of the University of Seville, and all experiments were in accordance with the recommendations of the European Eur J Nutr 1 3 Union regarding animal experimentation (Directive of the European Counsel 2010/630/EU). Induction of CIA Arthritis was induced in 10-week-old male DBA-1/J mice. Bovine type II collagen (CII) 2 mg/mL in dilute acetic acid (MD Bioproducts®, Zürich, Switzerland) was emulsified in equal volumes of Freund’s complete adjuvant (2 mg/ mL Mycobacterium tuberculosis, strain H37Ra; Difco®, Detroit, Michigan, USA). On day 0, DBA-1/J mice were immunized at the base of the tail with 100 mg of bovine CII. On day 21, mice received an intraperitoneal booster injection of 100 mg of CII dissolved in phosphate-buffered saline (PBS). Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Joint inflammation was scored visually in each paw, using a scale of 0–2 where 0 = uninflamed, 1 = mild, 1.5 = marked and 2 = severe. Scoring was performed by two independent observers without knowledge of the experimental groups. Animals were euthanized on day 42 by overdoses of pentobarbital. Histological analysis After mice were killed, knee joints were removed and fixed in 4 % formalin. After decalcification in 10 % EDTA, specimens were processed for paraffin embedding. Tissue sections (7 mm) were stained with hematoxylin and eosin (H&E) to perform histological analysis. Measurement of inflammatory markers Enzyme-linked immunoassay (ELISA) kits were used to determine serum levels of cartilage oligomeric matrix protein (COMP) levels, with sensitivity of 0.2 U/L (MD Biosciences®, Zurich, Switzerland) and matrix metalloproteinase-3 (MMP-3) (sensitivity of 10 pg/mL) (R&D Systems®, Abingdon, UK). Hind paws were amputated above the ankle and homogenized in 1 mL of 10 mM Table 1 Composition of experimental diets (g/kg diet) Diet was formulated on the basis of the AIN standard reference diet with the modification of various sources of carbohydrate a Sunflower oil from Koipesol® (Spain) b EVOO from picual variety, Oleoestepa®, Seville, Spain c Mineral mix provided the following (g/kg diet): calcium carbonate, 35.7; monopotassium phosphate, 25.0; sodium chloride, 7.4; potassium sulfate, 4.66; potassium citrate monohydrate, 2.8; magnesium oxide, 2.4; ferric citrate, 0.606; zinc carbonate, 0.165; manganese carbonate, 0.063; copper carbonate, 0.03; potassium iodate, 0.001; sodium selenate, anhydrous, 0.001025; ammonium molybdate-4H2O, 0.000795; sodium metasilicate-9H2O, 0.145; chromium potassium sulfate-12H2O, 0.0275; boric acid, 0.00815; sodium fluoride, 0.00635; nickel carbonate, 0.00318; lithium chloride, 0.00174; ammonium vanadate d Vitamin mix provided the following (g/kg diet): nicotinic acid, 30 mg; D-calcium pantothenate, 16 mg; pyridoxine HCL, 7 mg; thiamine HCL, 6 mg; riboflavin, 6 mg; folic acid, 2 mg; D-biotin, 0.2 mg; vitamin B12, 25 mg; α-tocopherol powder (250 U/g), 300 mg; vitamin A palmitate (250,000 U/mg), 16 mg; vitamin D3 (400,000 U/g), 2.5 mg; phylloquinone, 0.75 mg Ingredients SO diet EVOO diet Casein 200 200 DL-methionine 3 3 Corn starch 150 150 Sucrose 447.3 447.3 Cellulose 50 50 Sunflower oila100 – EVOOb– 100 Mineral mixc35 35 Vitamin mixd10 10 Choline bitarate 2 2 Table 2 Fatty acid, sterols, squalene, triterpene alcohols and tocopherol composition of EVOO and SO Parameters EVOO SO Fatty acid composition as determined by GC (% m/m methyl esters) Myristic C-14:0 0.01 0.08 Palmitic C-16:0 11.00 6.62 Palmitoleic C-16:1 0.90 0.12 Margaric C-17:0 0.10 0.04 Margaroleic C-17:1 0.10 0.03 Stearic C-18:0 3.10 4.27 Oleic C-18:1 79.40 24.83 Linoleic C-18:2 3.90 62.62 Linolenic C-18:3 0.70 0.17 Araquic C-20:0 0.40 0.27 Eicosenoic C-20:1 0.20 0.22 Behenic C-22:0 0.10 0.58 Lignoceric C-24:0 0.10 0.17 Sterols as determined by GC, relative amounts (%) Cholesterol 0.10 0.20 Brassicasterol 0.00 0.10 Campesterol 3.30 10.70 Stigmasterol 0.50 8.80 β-sitosterol 95.40 63.40 Δ7-Stigmastenol 0.40 10.50 Δ5-Avenasterol 0.00 6.2 Total sterols (mg/kg) 1,351 3,832 Squalene as determined by GC (mg/kg) 5,772 – Triterpene alcohols (mg/kg) 542 – Tocopherols and tocotrienols as determined by HPLC (mg/kg) α-Tocopherol (Vitamin E) 85 575 Eur J Nutr 1 3 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.4. Supernatants were used for the determination of IL-17, TNF-α and IL-1β levels, using the ELISA kit with sensitivities of 4.6 (Diaclone®, Besancon Cedex, France), 25 (Diaclone®, Besancon Cedex, France) and 5.1 (R&D Systems®, Abingdon, UK) pg/mL, respectively. Isolation of cytoplasmic and nuclear proteins and immunoblotting detection Frozen hind paws were homogenized in liquid N2. Isolation of cytoplasmic and nuclear proteins was performed as described by Rosillo et al. [25] with some modifications. Protein concentration of the homogenate was determined following Bradford’s colorimetric method [26]. Aliquots of supernatant that contains equal amount of protein (50 µg) were evaluated to determine HO-1 (Enzo®, Madrid, Spain), Nrf2, p-p38, p-JNK, p-STAT3, p65 (Santa Cruz Biotecnology®, CA, USA) and IκB-α (Cell Signalling Technology®, MA, USA) proteins by Western blot as described by Rosillo et al. [25]. The signals were analyzed and quantified by a scientific imaging systems (ImageJ software®, Maryland, USA). Data analysis All values in the figures and text are expressed as arithmetic mean ± standard error (SEM). Data were evaluated with Graph Pad Prism version 5.04 software. The statistical significance was evaluated by two-way (time course) or oneway (rest of parameters) analysis of variance (ANOVA), followed by Newman–Keuls’ test. P values < 0.05 were considered statistically significant. Results Effects of dietary EVOO on CIA model The development of arthritis was monitored until day 42. The time course of arthritic score (Fig. 1a) indicates that control mice fed with SO presented a progressive development of arthritis observed from day 29. However, mice feeding with EVOO showed a significant (p < 0.05 vs. SOCIA) delayed onset and decreased the disease severity of CIA reducing disease incidence, number of involved paws, footpad thickness and clinical index from days 30 to 42. These results suggested that dietary EVOO not only could retard the development but it also may have a therapeutic effect on ongoing inflammatory arthritis. Representative photographs of hind paws from the different experimental animal groups are shown in Fig. 1b. In addition, H&E staining revealed that histological features of the joint from sham animals were typical of normal structure with synovial membrane composed of synovial cells and collagen and a clear synovial space (Fig. 2a,b). On the contrary, joint from SO-CIA mice exhibited histological changes indicative of severe arthritis, characterized by an extensive infiltration of inflammatory cells into articular tissues, exudation into the synovial space, synovial hyperplasia and cartilage erosion (Fig. 2c). These histological features were less evident in EVOO-CIA group (Fig. 2d). Effects of dietary EVOO on serum biomarkers MMP-3 is well known as a predictor for joint destruction in early or established RA. In the present study, we determined serum MMP-3 levels, as synovial inflammatory biomarker, in CIA mice to investigate the potential beneficial effects of our nutritional therapeutic strategy. Circulating 28 30 32 34 36 38 40 42 44 0.0 0.4 0.8 1.2 1.6 2.0 2.4 SO-Sham SO-CIA EVOO-Sham ++++++ + + ** ** * *** ** EVOO-CIA Days Artritic Score A B cd ba Fig. 1 Time course of the arthritis macroscopic score (a), representative photographs (b) of hind paws at the end of the experiment (day 42). SO-Sham (a), non-arthritic mice feed with SO diet; EVOO-Sham (b), non-arthritic mice feed with EVOO diet; SO-CIA (c), control arthritic group feed with SO diet; EVOO-CIA (d), arthritic mice feed with EVOO diet. Data represent mean ± SEM n = 10. *p < 0.05 and **p < 0.01 vs Sham (SO and EVOO); +p < 0.05 vs SO-CIA Eur J Nutr 1 3 MMP-3 level was markedly increased (p < 0.001 vs. Sham) in serum from CIA animal group fed with SO diet (Fig. 3). By contrast, a strong reduction in MMP-3 serum levels was observed in those arthritic animals feeding with EVOO (p < 0.001 vs. SO-CIA) reaching levels comparable to those described in sham animals. In addition, serum levels of the cartilage degradation marker, COMP, were significantly elevated in SO-CIA animals when compared to sham controls. On the contrary, dietary EVOO significantly decreased serum COMP levels in CIA mice in comparison with those SO-CIA animals (p < 0.001 vs. SO-CIA) (Fig. 3). Effects of dietary EVOO on joint mediators It has been reported that TNF-α, IL-1β and IL-17 are critical cytokines involved in the pathogenesis of RA [27]. To explore whether cytokines joint levels were paralleled to the disease severity of CIA, concentration of these cytokines was examined in paw homogenates by ELISA. As shown in Fig. 4, TNF-α, IL-1β and IL-17 levels were significantly increased in paw homogenates from SO-CIA animals when compared with sham mice (p < 0.001 and p < 0.5 vs. Sham), suggesting its relationship with the synovial tissue inflammation. Conversely, our results indicate that animals fed with EVOO diet showed a significant reduction in all those pro-inflammatory cytokines production in comparison with SO-CIA group (TNF-α: p < 0,01, IL-1β: p < 0.001 and IL-17: p < 0.05 vs. SO-CIA). Importantly, IL-1β and IL-17 cytokines values were similar to those obtained from control sham animals. Effect of dietary EVOO on p-STAT3 protein expression STAT-3 has been described as a critical transcription factor involved in the pathogenesis of RA by steering the abnormal activation, automaticity and prolonged survival of synovial cells. Besides, STAT-3 is a key transcription factor involved in Th17 cell differentiation [28]. We evaluated p-STAT3 protein expression by Western blot from hind paw homogenates. Statistical analysis revealed a significant p-STAT-3 overexpression in SO-CIA group when compared to the sham control groups (p < 0.01 vs. Sham), whereas nutritional therapy with EVOO significantly suppressed STAT3 phosphorylation in arthritic CIA mice (p < 0.01 vs. SO-CIA) (Fig. 5). These results are in accordance with those obtained in the measurement of pro-inflam- matory cytokines levels suggesting that dietary EVOO may repress IL-17 production via negatively interfering with STAT3 signaling pathway. Fig. 2 Histological analysis of the frontal sections of knee joints on day 42. Sections were stained with hematoxylin and eosin. Original magnification x200. SO-Sham (a), non-arthritic mice feed with SO diet; EVOO-Sham (b), non-arthritic mice feed with EVOO diet; SO-CIA (c), control arthritic group feed with SO diet; EVOO-CIA. Arrows the infiltration of inflammatory cells and the cartilage erosion. d Arthritic mice feed with EVOO diet. The arrow less damage at the cartilage. The images are representative of at least six experiments Eur J Nutr 1 3 Effect of dietary EVOO on HO-1 and Nrf2 protein expression The expression of the proteins HO-1 and Nrf2 was also evaluated in paw homogenates by western blotting. Our data show that HO-1 was significantly downregulated in SO-CIA mice during the maintenance of chronic inflammation (p < 0.001 vs. Sham); however, dietary EVOO treatment induced a strong HO-1 overexpression in comparison with SO-CIA arthritic control group (p < 0.01 vs. SO-CIA) (Fig. 6). Nrf2 activation has been reported to play an important role in HO-1 expression. According to our results, nuclear Nrf2 expression was reduced in those arthritic animals feed with SO diet (p < 0.05 vs. Sham), whereas a significant increase in nuclear Nrf2 protein levels was observed in EVOO-CIA animals (p < 0.05 vs. SO-CIA) (Fig. 6). Effect of dietary EVOO on JNK and p38 MAPKs phosphorylation MAPKs play a key role inducing the pro-inflammatory gene expression, which initiates inflammatory responses. Sham CIA Sham CIA 0 2 4 6 ** +++ SO EVOO COMP (U/l) Sham CIA Sham CIA 0 50 100 150 200 250 *** +++ SO EVOO MMP-3 (ng/mL ) Fig. 3 Measurement of serum COMP and MMP-3 levels. These were measured by ELISA kits. Data represent mean ± SEM, n = 10. **p < 0.01 and ***p < 0.001 vs Sham (SO and EVOO); +++p < 0.001 vs SO-CIA Sham CIA Sham CIA 0 100 200 300 * + SO EVOO IL-17 (pg/g tissue ) Sham CIA ShamCIA 0 100 200 300 *** ++ SO EVOO TNF-α (pg/g tissue) Sham CIA Sham CIA 0 500 1000 1500 *** +++ SO EVOO IL-1β (pg/g tissue) Fig. 4 Measurement of TNF-α, IL-1β and IL-17 levels in hind paw homogenates. These were determined by ELISA kits. Data represent mean ± SEM, n = 10. *p < 0.05 and ***p < 0.001 vs Sham (SO and EVOO); +p < 0.05, ++p < 0.01 and +++p < 0.001 vs SO-CIA Eur J Nutr 1 3 We investigated the effect of dietary EVOO on MAPKs (JNK and p38) signaling pathway activation in CIA mice. In the present study, phosphorylation of JNK and p38 proteins increased in cytosolic extracts from SO-CIA mice paw homogenates. Nonetheless, the protein expression of both p-JNK and p-p38 was significantly ameliorated in EVOO-CIA mice (p < 0.05 and p < 0.01 vs. CIA-SO) (Fig. 7). Effects of dietary EVOO on NF-κB signaling pathway We also investigated the effects of EVOO on IkB-α degradation in cytoplasmic extracts from mice joints. As shown in Fig. 8, IκB-α expression was significantly reduced in CIA-induced arthritis mice when compared with sham mice, in the SO treatment. The IκB-α expression observed in CIA mice is consistent with an increase in IκB-α degradation, thus allowing NF-κB translocation into the nucleus to bind specific DNA sequences leading to pro-inflam- matory genes transcription. On the contrary, the effect observed in arthritic control group on IκB-α protein expression was prevented by dietary EVOO treatment. According to the results obtained, the nuclear p65 protein levels were increased in SO-CIA group (p < 0.001 vs. Sham), whereas dietary EVOO treatment prevented the CIA- induced nuclear translocation level of p65 in paw homogenate in comparison with those arthritic animals feed with SO (p < 0.01 vs. SO-CIA) (Fig. 8) avoiding the NF-κB- mediated transcriptional activation. Discussion The focus of this study has been to demonstrate the potential protective role of a diet elaborated with EVOO in the prevention and development of inflammatory arthritis and joint damage in a murine CIA-induced experimental arthritis model. This model is commonly used to investigate mechanisms relevant to RA as well as new antiarthritic treatments [10]. CIA induction resulted in the development of a pronounced synovitis associated with an autoimmune response against cartilage and production of matrix-degrad- ing enzymes accompanying cartilage degradation and bone erosions [29]. The results of the present work clearly indicate, for the first time, that EVOO, as the lipid component of the diet, effectively exhibited preventive and therapeutic effects in the development of inflammatory arthritis and joint damage in CIA arthritic mice in comparison with those CIA mice fed with SO. This effect was well correlated to an improved arthritis score, a minor inflammatory cells infiltration into articular tissues, reduced exudation into the synovial space, synovial hyperplasia and cartilage erosion. The pathogenesis of RA is closely related to an imbalance of cytokine network contributing to the development and progression of both CIA and RA [30]. Actually, overexpression of pro-inflammatory cytokines, such as IL-1β, Sham CIA Sham CIA 0.0 0.5 1.0 1.5 *** ++ SO EVOO Densitomery Analysis (Arbitrary units) Sham CIA Sham CIA SO EVOO Fig. 5 Changes in p-STAT3 protein expression in hind paws homogenate. The expression of p-STAT3 was quantified by densitometry and normalized with respect to β-actin. Data represent mean ± SEM, n = 4.***p < 0.001 vs Sham (SO and EVOO); ++p < 0.01 vs SO-CIA Sham CIA Sham CIA 0 1 2 3 4Nrf2 HO-1 SO EVOO *** * ++ + Densitometry Analysis (Arbitrary units ) Sham CIA Sham CIA SO EVOO Fig. 6 EVOO dietary up-regulated HO-1 and Nrf2 protein expression in hind paws homogenate. The expression was quantified by densitometry and normalized with respect to β-actin. Data represent mean ± SEM, n = 4.*p < 0.05 and ***p < 0.001 vs Sham (SO and EVOO); +p < 0.05 and ++p < 0.01 vs SO-CIA Eur J Nutr 1 3 TNF-α and IL-17, may activate osteoclasts and macrophages and also recruit leukocytes in inflamed joints. The infiltrated leukocytes produce cytokines, chemokines and tissue-destructive enzymes leading to chronic inflammation, cartilage damage and bone erosion. Besides, IL-17 is able to induce the release of IL-8 and IL-6, and plays a remarkable role in the additive/synergistic effects induced by TNF-α and IL-1β [31]. Importantly, targeting IL-17 has demonstrated therapeutic effects in animal models of inflammatory and autoimmune diseases, including RA [32]. Cytokines also up-regulate MMPs expression by chondrocytes and synoviocytes at the pannus cartilage junction leading to cartilage degradation [3, 4, 33]. Therefore, we examined TNF-α, IL-1β and IL-17 levels in paw homogenates from CIA animals in comparison with sham control group. Our results indicate that animals fed with EVOO diet showed a significant reduction in all pro-inflammatory cytokines levels. Consequently, EVOO intake would contribute to reduce the inflammatory response and joint damage by suppressing the production of the key Th17 polarization cytokines, TNF- α and IL-1β, in CIA arthritic mice. COMP is a matrix protein with a great potential as a biological marker of cartilage metabolism in arthritis [34]. Increased fragments of COMP have been reported in patients with osteoarthritis, RA and joint injury; thus, the monitoring of COMP levels in synovial fluid or serum has been suggested to be a helpful method for assessing the presence and progression of the inflammatory disease. In this sense, serum COMP levels are reduced in RA patients in remission [34, 35]. In addition, COMP is a putative substrate for MMPs. Particularly, MMP-3 is a proteinase secreted by synovial fibroblasts and chondrocytes. Its activity results in degradation of aggrecan core protein, cartilage link protein, fibronectin and collagen types IV, VII, IX and XI. MMP-3 is present in RA synovial fluid, overexpressed in rheumatoid synovium and then associated with higher joint damage in RA [36]. Likewise, serum MMP-3 level is suggested as a predictor for joint destruction in early RA or established RA and should be used in association with usual inflammatory markers to follow therapy efficiency [37]. Our data are in agreement with above studies and showed that high serum COMP and MMP-3 levels were associated with disease activity and joint progression in CIA mice; by contrast, the production of both cartilage and synovial biomarkers was significantly inhibited by dietary EVOO treatment in CIA mice. Abnormal signaling pathways play an important role in the inflammatory process and can lead to dysregulation of the inflammatory response being crucial in RA Sham CIA ShamCIA 0.0 0.5 1.0 1.5 p-p38 p-JNK *** ** ++ + SO EVOO Densitometry Analysi s (Arbitrary units ) Sham CIA Sham CIA SO EVOO Fig. 7 Effect of dietary EVOO on JNK and p38 MAPKs phosphorylation in hind paws homogenate. The expression of phosphorylated proteins was expressed related to the expression of the corresponding total protein. Data represent mean ± SEM, n = 4. **p < 0.01 and ***p < 0.001 vs Sham (SO and EVOO); +p < 0.05, ++p < 0.01 vs SO-CIA Sham CIA Sham CIA 0.0 0.2 0.4 0.6 0.8 1.0 IκB- αp65 *** ++ * + SO EVOO Densitometry Analysis (Arbitrary units ) Sham CIA Sham CIA SO EVOO Fig. 8 Expression of IκB-α in cytoplasmic fraction and p65 NF-κB in nuclear fraction of homogenates from hind paws. The expression was quantified by densitometry and normalized with respect to β-actin. Data represent mean ± SEM, n = 4. *p < 0.05 and ***p < 0.001 vs Sham (SO and EVOO); +p < 0.05 and ++p < 0.01 vs SO-CIA Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis☆ María Ángeles Rosillo a , María José Alcaraz b , Marina Sánchez-Hidalgo a , José G. Fernández-Bolaños c , Catalina Alarcón-de-la-Lastra a , María Luisa Ferrándiz b, ⁎ a Department of Pharmacology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain b Department of Pharmacology and IDM, University of Valencia, 46100 Burjasot, Valencia, Spain c Department of Organic Chemistry, Faculty of Chemistry, University of Seville, 41012 Seville, Spain Received 11 March 2014; received in revised form 30 June 2014; accepted 11 July 2014 Abstract The consumption of extra virgin olive oil (EVOO) in Mediterranean countries has shown beneficial effects. A wide range of evidence indicates that phenolic compounds present in EVOO are endowed with anti-inflammatory properties. In this work, we evaluated the effects of EVOO-polyphenol extract (PE) in a model of rheumatoid arthritis, the collagen-induced arthritis model in mice. On day 0, DBA-1/J mice were immunized with bovine type II collagen. On day 21, micereceiveda boosterinjection.PE(100and200mg/kg)wasorallyadministeredonceadayfromdays29to41toarthriticmice.WehavedemonstratedthatPEdecreasesjointedema, cell migration, cartilage degradation and bone erosion. PE significantly reduced the levels of proinflammatory cytokines and prostaglandin E 2 in the joint as well as the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1. Our data indicate that PE inhibits c-Jun N-terminal kinase, p38 and signal transducer and activator of transcription-3. In addition, PE decreases nuclear factor κB translocation leading to the down-regulation of the arthritic process. These results support the interest of natural diet components in the development of therapeutic products for arthritic conditions. © 2014 Elsevier Inc. All rights reserved. Keywords: Rheumatoid arthritis; CIA; EVOO; Polyphenols; Inflammatory response 1. Introduction Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and pannus leading to cartilage and bone damage. The earliest event is activation of the innate immune by exogenous and autologous antigens and thus auto-antibodies and later high levels of cytokines can be detected years before clinical symptoms [1]. At a joint level, there are synovial hyperplasia and massive infiltration of immune cells, including CD4 + T cells, B cells, natural killer cells, macrophages, dendritic cells, neutrophils and mast cells [2,3]. A wide range of evidence has demonstrated the contribution of cytokines to the chronicity of inflammation and tissue damage in RA. Therefore,the importance of proinflammatory cytokines has been confirmed by the success of cytokine-directed therapies in RA [4]. Proinflammatory cytokines such as interleukin (IL)-1βand tumor necrosis factor α(TNFα) activate many signal transduction pathways involved in inflammation. The mitogen-activated protein kinase (MAPK) pathway includes extracellular signal-regulated kinases (ERK1/2 or p42/p44), c-Jun N-terminal kinases (JNK)1/2/3 and p38, which are activated in the synovium of patients with RA [5].Thejanus kinase-signal transducer and activator of transcription (JAK-STAT) is another relevant inflammatory pathway activated in response to cytokines. In particular, overexpression of STAT-3 has been reported in synovial membranes from RA patients [6]. Proinflammatorycytokinesactivatedifferenttranscriptionfactorsto induce the expression of inflammatory and catabolic mediators [7]. Nuclear factor κB(NF-κB) and activating protein-1 (AP-1) are activated in RA synovium and play an important role in metalloproteinase induction [8].NF-κB regulates a wide range of genes that contribute to inflammation, such as IL-1β,TNFα, IL-6, chemokines and microsomal prostaglandin E synthase-1 (mPGES-1), an efficient downstream enzyme functionally coupled with cyclooxygenase-2 (COX-2). Biological therapies have improved the treatment of chronic inflammatory diseases such as RA. Nevertheless, these drugs are effective in a fraction of patients only and have other limitations including a high cost, the requirement for parenteral administration and important side effects. Therefore, new therapeutic strategies are under investigation. Epidemiological studies about consumption of functional foods, particularly extra virgin olive oil (EVOO) in Mediterranean countries, have showed important beneficial effects. Available online at www.sciencedirect.com ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 1275–1281 ☆ Supported by research grants SAF2010-22048, RETICEF RD12/0043/ 0013and AGL 2011-26949 (Ministeriode Economía y Competitividad,ISCIII, FEDER), PROMETEO II 2014-071 (Generalitat Valenciana) and P-10AGR- 6609 (Junta de Andalucía). ⁎Corresponding author. Department of Pharmacology, Faculty of Pharmacy, Av.VicentAndrésEstelléss/n,46100Burjasot,Valencia,Spain.Tel.: +34 963543284. E-mail address: [email protected] (M.L. Ferrándiz). http://dx.doi.org/10.1016/j.jnutbio.2014.07.006 0955-2863/© 2014 Elsevier Inc. All rights reserved. EVOO, obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to oil alteration, has shown antioxidant, anti-inflammatory, immunomodulatory, antiproliferative and anti-apoptotic effects [9–15]. Although these health-protective effects have been traditionally ascribed to its high monounsaturated fatty acid content [15,16] a wide range of evidence indicates that many of the beneficial effects of EVOO intake are due to its minor highly bioactive components. Among them, phenolic compounds such as hydroxytyrosol, tyrosol and oleuropein have shown anti-inflammatory and antioxidant effects [17]. Olive oil polyphenols have also been associated with neuroprotective, antiaging and antiatherogenic effects and the regulation of important cellular signaling pathways. On the other hand, because of their ability to modulate cell death, olive polyphenols have been proposed as chemopreventive and therapeutic anti-cancer agents [18–21].Our research group has previously demonstrated that a diet made with EVOO enriched with hydroxytyrosol, could improve inflammatory processes in a chronic colitis model [22]. In addition, we evaluated the protective effect of dietary EVOO-polyphenol extract (PE) supplementation in the inflammatory response associated to a chronic colitis model [23]. Collagen-induced arthritis (CIA) in DBA/1 mice is an animal model of RA widely used to test potential therapeutic agents [24]. The pathogenesis of CIA is dependent on the host's response to type II collagen challenge and the subsequent generation of antibodies that recognize collagen-rich joint tissue [25]. Joint inflammation in this model is characterized by significant cellular exudate and infiltrate, synovitis, and articular degradation. High levels of proinflammatory cytokines are detected at the onset of the disease, leading to the production of a wide range of mediators relevant in CIA pathogenesis [26,27]. Taken into account the anti-inflammatory properties of olive oil constituents, we hypothesized that PE may control the development of inflammation and articular lesion. In fact, intraperitoneal administration of oleuropein aglycone, a majorconstituentof the leaves and unprocessed olive drupes, has shown beneficial effects in CIA [28], and a recent study has reported that EVOO diet in conjunction with physical activity is able to preserve the articular cartilage in an osteoarthritis model [29]. The present study was designed to evaluate the effects of the oral administration of PE in the arthritis model of CIA in mice. In addition to macroscopic and histological analyses, we have determined the effects of PE on the production of inflammatory mediators. In order to gain a better insight into its mechanisms of action, signaling pathways were also explored. 2. Materials and methods 2.1. Extraction of PE PE was obtained by the method described by Vazquez Roncero et al. [30] with some modifications [31]. Fifty grams of EVOO (Oleoestepa, Seville, Spain) was extracted with methanol/water (80:20, vol/vol, 125 ml). The mixture was mixed with a vortex at 5000 g for 1 min and sonicated for 15 min. After decantation, the methanolic extract was concentrated in vacuum under a stream of nitrogen at b35°C until it reached a syrup consistency; finally, it was lyophilized and stored at −80°C. 2.2. Characterization of phenolic compounds in PE PE (70–75 mg) extracted from EVOO (75 g) was dissolved in CDCl 3 (750 μl) or in DMSO-d6 (750 μl) and an precisely measured volume of the solution (550 μl) was placed in a 5 mm NMR tube for the detection and quantification of phenolic compounds. The phenolic mixture to be analyzed in DMSO-d6 was previously dissolved in a MeOD-D 2 O 1:1 (1 ml) and concentrated to dryness at reduced pressure in order to exchange hydroxyl protons with deuterium nuclei. The NMR solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). NMR experiments were conducted on a Bruker Avance III 700 spectrometer, operating at 700.25 M Hz. The probe temperature was 24.8±1°C. All chemical shifts were given in ppm, and the J couplings in Hz. The solvent itself was used as a chemical shift reference (7.26 ppm for CHCl 3 , and 2.50 for DMSO-d6). 1 H NMR spectra were acquired with the following acquisition parameters: acquisition time 2.3s, relaxation delay 5s, spectral width of 0–20 ppm, data points 32k, number of scans 32, and line broadening of 0.3 Hz. Postacquisition processing included zero-filling to 64k. The spectra were phase corrected automatically using TOPSPIN and integration was performed manually. We have determined the levels of oleocanthal and oleacein following a modification of the method developed by Karkoula et al. [32] for direct measurement of both dialdehydes in olive oil by quantitative high resolution 1 H NMR in CDCl 3 . The levels of other phenolic compounds have been determined by the procedure described by Christophoridou and Dais [33]. 2.3. Induction of CIA Arthritis was induced in male DBA-1/J mice (Janvier, Le Genest St Isle, France) of 11 weeks of age. All studies were performed in accordance with European Union regulations for the handling and use of laboratory animals. The protocols were approved by the institutional Animal Care and Use Committee (University of Valencia, Spain). Bovine type II collagen (CII; 2 mg/ml in dilute acetic acid; MD Biosciences, Zürich, Switzerland) was emulsified in equal volumes of Freund's complete adjuvant (2 mg/ml Mycobacterium tuberculosis, strain H37Ra; Difco, Detroit, MI, USA). On day 0, DBA-1/J mice were immunized at the base of the tail with 100 µg of bovine CII. On day 21, mice received an intraperitoneal booster injection of 100 µg of CII dissolved in phosphate-buffered saline. Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Joint inflammation was scored visually in each paw, using a scale of 0–2 where 0=uninflamed, 1=mild, 1.5=marked and 2=severe. Scoring was performed by two independent observers without knowledge of the experimental groups. In addition, photographs and X-ray (Carestream MS FX, Gainesville, FL, USA) images of hind paws were obtained at the end of the experiment (day 42). 2.4. Treatment groups On day 29, animals were randomized into control and treatment groups. The study was performed in four groups of mice (n=10): naive group (NA), CIA group (CIA) and two treatment groups: CIA mice with PE treatment (PE 100 and 200 mg/kg, orally, once a day from days 29 to 41). On day 42 after immunization mice were anesthetized, blood samples were taken by intracardiac puncture and animals were killed by cervical dislocation. Paws were amputated and processed for either histological analysis or homogeneization and assessment of inflammatory mediators and mechanisms. 2.5. Histological and immunohistochemical analyses Knee joints were fixed in 4% formalin. After decalcification in 10% EDTA, specimens were processed for paraffin embedding. Tissue sections (7 μm) were stained with hematoxylin and eosin to perform histological analyses. For immunohistochemistry, the endogenous peroxidase activity was inhibited with hydrogen peroxide and then the sections were incubated in normal horse serum (Vectastain Kit; Vector Laboratories, Burlingame, CA, USA) for 20 min to reduce nonspecific staining and successively incubated with rabbit anti-COX-2 (Cayman Chemical, Ann Arbor, MI, USA) at dilution 1:250 overnight at 4°C. Later on, slides were treated with antirabbit IgG antibody (Vectastain Kit, Vector Laboratories, Burlingame, CA, USA) for 30 min and incubated with the streptavidin–peroxidase complex of the kit for 30 min, at room temperature. After incubation with the peroxidase substrate 3,3′-diaminobenzidine and washing with water, the sections were counterstained with hematoxylin. Negative control sections were treated in the same way but in the absence of primary antibody. Positive cells and total cells were counted in five random high-power fields by two independent observers. 2.6. Enzyme-linked immunosorbent assay Hind paws (knees) were homogenized in liquid N 2 with 1 ml of A buffer pH 7.46 (10 mM HEPES, pH 8, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol (DTT), 5 mM NaF, 1 mM Na 3 VO 4 ,1μg/ml leupeptin, 0.1 μg/ml aprotinine, and 0.5 mM phenylmethyl sulfonyl fluoride (PMSF)). The tissue homogenates were sonicated (10 s three times at 20% with a 10-s incubation on ice between bursts) in an ultrasonic processor (VC130PB, Sonics & Materials Inc., Newtown, CT, USA) and centrifuged at 600×g, 15 min at 4°C. Supernatants were removed and used for determination of inflammatory mediators by enzyme-linked immunosorbent assay (ELISA): PGE 2 (Cayman Chemical, Michigan, USA), TNFαand IL-1β(R&D Systems Inc., Minneapolis, MN, USA), and IL-6 (Diaclone, Besançon Cedex, France). 2.7. Western blot Hind paws were homogenized in liquid N 2 with 1 ml of ice-cold phosphate buffer (0.01 mM K 2 HPO 4 ,KH 2 PO 4 and 0.15 M NaCl) and centrifuged (800×g,10 min at room temperature). Pellets were resuspended in hypotonic buffer [1.5 mM MgCl 2 ,10mMKCl,0.2mMPMSF,1μg/ml leupeptin, 20 mM NaF, 0.5 mM DTT, 1 mM EDTA and 10 mM HEPES, pH 7.9], incubated for 10 min on ice and centrifuged (14,000×g, 30 s at room temperature). Supernatants (cytoplasmic proteins) and pellets (nuclear proteins) were collected. Pellets were resuspended in ice-cold low-salt buffer and nuclear proteins were released by adding a 1276 M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 high-salt buffer(20%vol/volglycerol,1.5mMMgCl 2 , 0.2 mM EDTA,0.2 mMPMSF, 0.5 mM DTT, 420 mM NaCl, 0.2 mM EDTA, 20 mM NaF, 1 μg/ml leupeptin and 20 mM HEPES, pH 7.9). Samples were incubated on ice for 30 min. Soluble nuclear proteins were recovered by centrifugation (14,000×g, 10 min, 4°C) and proteins were stored at −80°C. Protein concentration was determined by the Bradford's colorimetric method [34]. Nuclear fractions were used to study p65 protein expression and cytoplasmic fractions were used for the rest of determinations. Aliquots containingequal amount of protein (50 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel). The proteins were then electrophoretically transferred onto a nitrocellulose membrane and incubated with specific primary antibodies: rabbit polyclonal anti-mPGES-1 (1:200; Cayman Chemical, USA) rabbit polyclonal anti-IκB-α(1:1000; Cell Signalling Technology, USA),rabbit polyclonal p65 (1:200, SantaCruzBiotechnology, Inc.), rabbitpolyclonalantip-JNK and rabbit polyclonal anti-JNK (1:1000; Santa Cruz Biotechnology, Inc.), mouse polyclonal anti-p-p38 and rabbit polyclonal anti-p38 (1:1000; Santa Cruz Biotechnology, Inc.) and mouse polyclonal anti-p-STAT3 (1:200; Santa Cruz Biotechnology, Inc.), overnight at 4°C. Membranes were washed three times for 15 min and incubated with anti-rabbit (Pierce Biotechnology, IL, USA) or anti-mouse (Dako, Carpinteria, CA, USA) horseradish peroxidase-labeled secondary antibody, for 1–2 h at room temperature. To prove equal loading, the blots were analyzed for β-actin expression using an anti-β-actin antibody (Sigma–Aldrich). Immunodetection was performed using enhanced chemiluminescence light-detecting kit (SuperSignal West Femto Chemiluminescent Substrate, Pierce, IL, USA). Densitometric data were studied following normalization to the control (housekeeping gene). The signals were analyzed and quantified by ImageJ software (Bethesda, MD, USA). 2.8. Data analysis All values in the figures and text are expressed as arithmetic means±standard error (S.E.M.). Data were evaluated with Graph Pad Prism Version 5.04 software. The statistical significance was evaluated by two-way (time course) or one-way (rest of parameters) analysis of variance (ANOVA), followed by Bonferroni's test. Pvalues b.05 were considered statistically significant. 3. Results 3.1. Chemical composition of PE Table 1 shows the result of qualitative and quantitative analyses of PE. Nine different phenolic compounds have been identified. Oleocanthal and oleacein are the main components of PE. 3.2. Effect of PE on established CIA The time-course of arthritic score (Fig. 1A) indicates that control CIA mice showed a progressive development of clinical symptoms. The severity of arthritis in the groups treated with PE (100 and 200 mg/kg) was lower than that of the arthritic control group with significant results for the highest dose on days 39 and 41. Fig. 1B shows representative photographs of hind paws from the different groups. In addition, X-ray images (Fig. 1C) revealed the presence of joint edema and focal bone erosions in hind paws of CIA mice, whereas in animals treated with PE, the inflammation was lower. Interestingly, in the PE200 group, the integrity of bone was similar to that of naïve mice. Control CIA mice exhibited histological changes indicative of severe arthritis, with extensive infiltration of inflammatory cells into articular tissues, exudation into the synovial space, synovial hyperplasia and cartilage erosion (Fig. 2). These histological features were less apparent in mice treated with PE (200 mg/kg). 3.3. Effect of PE on joint inflammatory mediators We also explored whether PE could modify the production of proinflammatory cytokines and PGE 2 . At the end of the experimental period (day 42), the levels of IL-1β, TNFαand IL-6 were determined in hind paw homogenates. As shown in Fig. 3, animals treated with PE (100 or 200 mg/kg) showed a significant reduction in joint levels of all proinflammatory cytokines. In addition, a significant increase in PGE 2 levels in the paws of arthritic control animals with respect to naïve mice was found (Fig. 3). Treatment with PE reduced the levels of this eicosanoid, which reached statistical significance at both doses. 3.4. Effect of PE on COX-2 and mPGES-1 expression The effect of PE on COX-2 protein expression was determined by immunohistochemistry of knee joint sections (Fig. 4A). We observed a significant increase in the percentage of COX-2-positive cells in cartilage of arthritic control group (CIA), with respect to naïve animals. In the group treated with PE (200 mg/kg), the immunoreactivity for this protein was significantly reduced. COX-2 expression was accompanied by a significant increase in mPGES-1 protein expression, which was determined by Western blot, in the paws of CIA control mice with respect to naïve animals (Fig. 4B). Treatment with PE significantly decreased the expression of this protein. 3.5. Effect of PE on p38 and JNK phosphorylation p38 and JNK play an important role in inflammation and tissue damage in RA [5,35]. We investigated the effect of PE on the activation of JNK and p38 in the CIA model by western blot using phosphospecific antibodies. The development of CIA induced strong MAPK phosphorylation in joints (Fig. 5). However, PE at both doses significantly decreased the phosphorylation of JNK and p38. 3.6. Effect of PE on p-STAT3 protein expression in paw homogenate p-STAT3 protein expression was evaluated by Western blot analysis of hind paw homogenates. Fig. 6 shows that arthritis strongly induced STAT3 phosphorylation, as observed in CIA control mice with respect to naïve animals. Treatment with PE at 200 mg/kg and, to a lower extent, at 100 mg/kg, significantly reduced the expression of p- STAT3 in arthritic mice. 3.7. Effects of PE on NF-κB signaling pathway We investigated the effect of PE on IκB-αexpression in cytoplasmic extracts of mice joints. As shown in Fig. 7, the expression of IκB-αwas significantly reduced in arthritic control mice (CIA) compared with naïve mice. The effect of arthritis on IκB-αwas blocked by PE treatment at the dose of 200 mg/kg. The reduction in IκB-αexpression observed in CIA mice is consistent with an increase in IκB-αdegradation thus allowing the translocation of NF-κB into the nucleus to bind specific DNA sequences leading to the transcription of proinflammatory genes. Therefore, CIA mice showed an increased expression of p65 NF-κB in nuclear extracts of joints which was significantly inhibited by PE at 200 mg/kg. 4. Discussion In this study, we have demonstrated that oral administration of PE is able to down-regulate the arthritic process in the CIA model of RA. PE showed anti-inflammatory effects with reductions in joint edema and migration of inflammatory cells. In addition, PE protected joints against cartilage alterations and bone erosion. According to previous Table 1 Chemical constituents of PE Phenolic compound μmol/100 g ppm Free hydroxytyrosol 2.25 3.47 Free tyrosol 5.21 7.20 Oleacein 9.62 30.82 Oleocanthal 10.43 31.74 Oleuropein aglycone monoaldehyde (5R,8S,9R) 1.49 5.63 Ligstroside aglycone monoaldehyde (5R,8S,9R) 3.88 14.06 Apigenin 0.56 1.52 Luteolin 1.26 3.61 Acetoxypinoresinol 1.92 8.00 1277M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 reportsonthebiologicalproperties ofoliveoilconstituents[17,18,28,9], the anti-inflammatory effects of PE are likely dependent on the mixture of different phenolic compounds. The progression of arthritis is associated with sustained production of proinflammatory cytokines [4]. We have shown in the CIA model that PE controls the local levels of these cytokines driving the production of different inflammatory and catabolic mediators. Proinflammatory cytokines induce further cytokines, chemokines, eicosanoids and reactive oxygen species (ROS) amplifying the inflammatory response [36]. Therefore, COX-2 and mPGES-1, enzymes responsible for the overproduction of PGE 2 in inflammation, are up-regulated [37] contributing to the progression of RA through EP 4 receptor activation [38]. We have shown that PE reduces PGE 2 levels which would be dependent on the down-regulation of COX-2 and mPGES-1 expression in the joint. Cytokines also induce the expression of matrix metalloproteinases at the pannus cartilage junction leading to cartilage degradation [39,40]. In addition, TNFα stimulates osteoclastogenesis [41], suppresses the recruitment of osteoblasts and inhibits the expression of matrix genes [42], whereas IL-6 increases osteoclast numbers in trabecular bone [43]. Therefore, inhibition of proinflammatory cytokine productionby PE would result in the reduction of the inflammatory response and tissue damage. MAPKs regulate the synthesis of chemokines, cytokines, adhesion molecules and PGs involved in inflammation. In addition, they mediate the induction of matrix metalloproteinases responsible for cartilage breakdown. JNK1/2 and p38 are also involved in osteoclast differentiation and thus the activation of these enzymes may contribute to bone destruction [35]. In particular, p38 plays a key 0 1 2 3 4 29 31 34 36 39 41 A Days Naïve CIA PE100 PE200 Clinical score ** * ++ ++ ++ Naïve CIA PE100 PE200 BC Naïve CIA PE100 PE200 Fig. 1. Time course of the arthritis macroscopic score (A), representative photographs (B) and X-ray images (C) of hind paws at the end of the experiment (day 42). Naïve, non-arthritic mice;CIA,controlarthriticgroup;PE 100,arthriticmicetreatedwithPE(100mg/kg/dayp.o.fromdays29to41 afterimmunization);PE 200,arthritic micetreatedwithPE(200mg/kg/day p.o. from days 29 to 41 after immunization). Data represent mean±S.E.M. n=10. +Pb.05, ++Pb.01 vs. naïve; *Pb.05, **Pb.01 vs. CIA. Arrows indicate focal bone erosion. Naïve CIA PE 200 Fig. 2. Histological analysis of the frontal sections of knee joints on day 42. Sections were stained with hematoxylin and eosin. Original magnification ×100. Naïve, nonarthritic mice; CIA, control arthritic group; PE 200, arthritic mice treated with PE (200 mg/kg/day p.o. from days 29 to 41 after immunization). The images are representative of at least six experiments performed on different days. 1278 M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 role in RA [44]. JAKs bind to intracellular domains of IL-6 or interferon receptors and catalyze ligand-induced phosphorylation of themselves and of intracellular tyrosine residues. Phosphorylation of STATs on tyrosine residues results in the formation of STAT dimers that translocate into the nucleus to bind specific DNA sequences. This pathway can be regulated by different protein kinases such as MAPK that phosphorylate STATs on serine residues to potentiate STAT-activating stimuli [45]. STAT3 may contribute to the chronicity of inflammation in experimental arthritis [46]. Our data indicate that PE administration is able to reduce the phosphorylation of JNK, p38 and STAT3, suggesting that PE could control the activation of these important signaling pathways during the arthritic process. The NF-κB pathway is involved in the transcription of many inflammatory genes [47]. Cellular stimulation by proinflammatory cytokines induces the recruitment of costimulatory molecules, such as TNF receptor-associated factor (TRAF) leading to the activation of NF-κB-inducing kinase. This protein induces IκB kinase activation resulting in the phosphorylation of IκB[6], which is followed by ubiquitination and proteolytic degradation thus allowing the release of NF-κB to enter the nucleus and regulate gene transcription [48]. 0 1000 2000 3000 4000 5000 0 20 40 60 80 0 1000 2000 3000 4000 ++ ** ** IL-1β pg/g tissue ++ ** ** pg/g tissue TNFα ++ *** pg/g tissue IL-6 Naïve CIA PE 100 PE 200 Naïve CIA PE 100 PE 200 Naïve CIA PE 100 PE 200 0 10 20 30 40 Naïve CIA PE 100 PE 200 PGE2 ng/g tissue ++ ** * Fig. 3. Levels of TNFα, IL-1β, IL-6 and PGE 2 in paw homogenates. These inflammatory mediators were determined by ELISA. Data represent mean±S.E.M., n=6. ++Pb.01 vs. naïve; *Pb.05, **Pb.01 vs. CIA. BA 0 20 40 60 80 100 ++ ** Naïve CIA PE 200 % Positive cells COX-2 0.0 0.2 0.4 0.6 0.8 1.0 Naïve CIA PE 200 Naïve CIA PE 100 PE 200 Naïve CIA PE 100 PE 200 ++ ** ** mPGES-1 mPGES-1/β-actin Fig. 4. Expression of COX-2 (A) and mPGES-1 (B) in hind paws. COX-2 was determined by immunohistochemistry and expressed as percentage of positive cells. mPGES-1 was determined by Western blot, quantified by densitometry and normalized with respect to β-actin. Data represent mean±S.E.M., n=4. ++Pb.01 vs. naïve; **Pb.01 vs. CIA. 1279M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 Our results indicate that PE enhances the levels of the inhibitory protein IκB-αleading to the reduction of NF-κB nuclear translocation. This mechanism may be responsible for the down-regulation of proinflammatory cytokines in the joint during the CIA process in the animals treated with PE. Proinflammatory cytokines and ROS could act as mediators of NF-κB activation [49]. As demonstrated in previous studies, PE inhibits the generation of ROS [50] besides its effect on proinflammatory cytokines, suggesting that both mechanisms could be involved in the control of NF-κB translocation. In conclusion, or study has demonstrated the anti-inflammatory and joint protective effects of PE from EVOO, which would be related to the inhibition of relevant signaling pathways such as NF-κB, STAT- 3, JNK and p38 controlling the production of inflammatory mediators. Our data also show the interest of natural diet components in the development of therapeutic products for arthritic conditions. Acknowledgment M.A.R. and M.L.F. performed the experiments and data analysis. M.S.H. and J.G.F.B. performed part of the analysis. C.A. and M.J.A. designed the study and prepared the manuscript. All authors read and approved the final content of the manuscript. The authors declare that they have no conflicts of interest.The authors gratefully acknowledge the assistance of Center for Technology and Innovation Research, University of Seville (CITIUS) and the I+D+i Oleoestepa SAC Department who gave us the EVOO. References [1] Jorgensen KT, Wiik A, Pedersen M, Hedegaard CJ, Vestergaard BF, Gislefoss RE, et al. Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case–control study nested in a cohort of Norwegian blood donors. Ann Rheum Dis 2008;67:860–6. [2] Tak PP, Bresnihan B. The pathogenesis and prevention of joint damage in rheumatoid arthritis: advances from synovial biopsy and tissue analysis. Arthritis Rheum 2000;43:2619–33. [3] Komatsu N, Takayanagi H. Inflammation and bone destruction in arthritis: synergistic activity of immune and mesenchymal cells in joints. Front Immunol 2012;3:77. [4] Goronzy JJ, Weyand CM. Developments in the scientific understanding of rheumatoid arthritis. Arthritis Res Ther 2009;11:249. [5] Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12. [6] Morel J, Berenbaum F. Signal transduction pathways: new targets for treating rheumatoid arthritis. Joint Bone Spine 2004;71:503–10. [7] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–61. Naïve CIA PE 100 PE 200 0.0 0.2 0.4 0.6 0.8 1.0 p38 JNK ++ ++ ** ** ** * Phosphorylated/total Naïve CIA PE 100 PE 200 Fig. 5. Expression of p-p38 and p-JNK in hind paws. The expression of phosphorylated proteins was expressed related to the expression of the corresponding total protein. Data represent mean±S.E.M., n=4. ++Pb.01 vs. naïve; *Pb.05, **Pb.01 vs. CIA. 0.0 0.2 0.4 0.6 0.8 1.0 Naïve CIA PE 100 PE 200 Naïve CIA PE 100 PE 200 ++ * ** p-STAT3/β-actin Fig. 6. Expression of p-STAT3 in hind paws. The expression of p-STAT3 was quantified by densitometry and normalized with respect to β-actin. Data represent mean±S.E.M., n=4. ++Pb.01 vs. naïve; *Pb.05, **Pb.01 vs. CIA. 0.0 0.5 1.0 1.5 2.0 Naïve CIA PE 100 PE 200 ++ ++ ** ** IκB-α p65 Naïve CIA PE 100 PE 200 Protein/β-actin Fig. 7. Expression of IκBαin cytoplasmic fraction and p65 NF-κB in nuclear fraction of homogenates from hind paws. The expression was quantified by densitometry and normalized with respect to β-actin. Data represent mean±S.E.M., n=4. ++Pb.01 vs. naïve; **Pb.01 vs. CIA. 1280 M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 [8] Han Z, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-kappaB regulation in rheumatoid arthritis and murine collagen-induced arthritis. Autoimmunity 1998; 28:197–208. [9] Rosignoli P, Fuccelli R, Fabiani R, Servili M, Morozzi G. Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes. J Nutr Biochem 2013;24:1513–9. [10] Bullo M, Lamuela-Raventos R, Salas-Salvado J. Mediterranean diet and oxidation: nuts and olive oil as important sources of fat and antioxidants. Curr Top Med Chem 2011;11:1797–810. [11] Scotece M, Gomez R, Conde J, Lopez V, Gomez-Reino JJ, Lago F, et al. Oleocanthal inhibits proliferation and MIP-1alpha expression in human multiple myeloma cells. Curr Med Chem 2013;20:2467–75. [12] Servili M, Esposto S, Fabiani R, Urbani S, Taticchi A, Mariucci F, et al. Phenolic compounds in olive oil: antioxidant, health and organoleptic activities according to their chemical structure. Inflammopharmacology 2009;17:76–84. [13] Sanchez-Fidalgo S, Villegas I, Cardeno A, Talero E, Sanchez-Hidalgo M, Motilva V, et al. Extra-virgin olive oil-enriched diet modulates DSS-colitis-associated colon carcinogenesis in mice. Clin Nutr 2010;29:663–73. [14] Oliveras-Ferraros C, Fernandez-Arroyo S, Vazquez-Martin A, Lozano-Sanchez J, Cufi S, Joven J, et al. Crude phenolic extracts from extra virgin olive oil circumvent de novo breast cancer resistance to HER1/HER2-targeting drugs by inducing GADD45-sensed cellular stress, G2/M arrest and hyperacetylation of Histone H3. Int J Oncol 2011;38:1533–47. [15] Escrich E, Solanas M, Moral R, Escrich R. Modulatory effects and molecular mechanisms of olive oil and other dietary lipids in breast cancer. Curr Pharm Des 2011;17:813–30. [16] Bermudez B, Lopez S, Ortega A, Varela LM, Pacheco YM, Abia R, et al. Oleic acid in olive oil: from a metabolic framework toward a clinical perspective. Curr Pharm Des 2011;17:831–43. [17] Omar SH. Oleuropein in olive and its pharmacological effects. Sci Pharm 2010;78: 133–54. [18] Visioli F, Bernardini E. Extra virgin olive oil's polyphenols: biological activities. Curr Pharm Des 2011;17:786–804. [19] Corona G, Spencer JP, Dessi MA. Extra virgin olive oil phenolics: absorption, metabolism, and biological activities in the GI tract. Toxicol Ind Health 2009;25:285–93. [20] Cardeno A, Sanchez-Hidalgo M, Alarcon-de-la-Lastra C. An up-date of olive oil phenols in inflammation and cancer: molecular mechanisms and clinical implications. Curr Med Chem 2013;20:4758–76. [21] Bernini R, Merendino N, Romani A, Velotti F. Naturally occurring hydroxytyrosol: synthesis and anticancer potential. Curr Med Chem 2013;20:655–70. [22] Sanchez-Fidalgo S, Sánchez de Ibargüen L, Cardeno A, Alarcon dlL. Influence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model. Eur J Nutr 2012;51:497–506. [23] Sanchez-Fidalgo S, Cardeno A, Sanchez-Hidalgo M, Aparicio-Soto M, de la Lastra CA. Dietary extra virgin olive oil polyphenols supplementation modulates DSS- induced chronic colitis in mice. J Nutr Biochem 2013;24:1401–13. [24] Wooley PH. Collagen-induced arthritis in the mouse. Methods Enzymol 1988;162: 361–73. [25] Holmdahl R, Andersson M, Goldschmidt TJ, Gustafsson K, Jansson L, Mo JA. Type II collagen autoimmunity in animals and provocations leading to arthritis. Immunol Rev 1990;118:193–232. [26] Mauri C, Williams RO, Walmsley M, Feldmann M. Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur J Immunol 1996;26:1511–8. [27] Maicas N, Ibañez L, Alcaraz MJ, Ubeda A, Ferrandiz ML. Prostaglandin D2 regulates joint inflammation and destruction in murine collagen-induced arthritis. Arthritis Rheum 2012;64:130–40. [28] Impellizzeri D, Esposito E, Mazzon E, Paterniti I, Di PR, Morittu VM, et al. Oleuropein aglycone, an olive oil compound, ameliorates development of arthritis caused by injection of collagen type II in mice. J Pharmacol Exp Ther 2011;339:859–69. [29] Musumeci G, Trovato FM, Pichler K, Weinberg AM, Loreto C, Castrogiovanni P. Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression. J Nutr Biochem 2013;24:2064–75. [30] Vazquez Roncero A, Janer del Valle M, Janer del Valle L. Componentes fenolicos de la aceituna. III. Polifenoles del aceite. Grasas Aceites 1976;27:185–90. [31] Allouche Y, Jimenez A, Gaforio JJ, Uceda M, Beltran G. How heating affects extra virgin olive oil quality indexes and chemical composition. J Agric Food Chem 2007;55:9646–54. [32] Karkoula E, Skantzari A, Melliou E, Magiatis P. Direct measurement of oleocanthal and oleacein levels in olive oil by quantitative (1)H NMR. Establishment of a new index for the characterization of extra virgin olive oils. J Agric Food Chem 2012; 60:11696–703. [33] Christophoridou S, Dais P. Detection and quantification of phenolic compounds in olive oil by high resolution 1H nuclear magnetic resonance spectroscopy. Anal Chim Acta 2009;633:283–92. [34] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [35] Thalhamer T, McGrath MA, Harnett MM. MAPKs and their relevance to arthritis and inflammation. Rheumatology (Oxford) 2008;47:409–14. [36] Abramson SB, Amin A. Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage. Rheumatology (Oxford) 2002;41:972–80. [37] Lazarus M, Kubata BK, Eguchi N, Fujitani Y, Urade Y, Hayaishi O. Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages. Arch Biochem Biophys 2002;397:336–41. [38] McCoy JM, Wicks JR, Audoly LP. The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J Clin Invest 2002;110:651–8. [39] Ribbens C, Porras M, Franchimont N, Kaiser MJ, Jaspar JM, Damas P, et al. Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis 2002;61:161–6. [40] Okada Y, Nagase H, Harris Jr ED. Matrix metalloproteinases 1, 2, and 3 from rheumatoid synovial cells are sufficient to destroy joints. J Rheumatol 1987;14 [Spec No:41–2]. [41] Cenci S, Weitzmann MN, Roggia C, Namba N, Novack D, Woodring J, et al. Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha. J Clin Invest 2000;106:1229–37. [42] Nanes MS. Tumor necrosis factor-alpha: molecular and cellular mechanisms in skeletal pathology. Gene 2003;321:1–15. [43] Jilka RL, Hangoc G, Girasole G, Passeri G, Williams DC, Abrams JS, et al. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 1992;257:88–91. [44] Hammaker D, Firestein GS. “Go upstream, young man”: lessons learned from the p38 saga. Ann Rheum Dis 2010;69(Suppl 1):i77–82. [45] Aaronson DS, Horvath CM. A road map for those who don't know JAK-STAT. Science 2002;296:1653–5. [46] de Hooge AS, van de Loo FA, Koenders MI, Bennink MB, Arntz OJ, Kolbe T, et al. Local activation of STAT-1 and STAT-3 in the inflamed synovium during zymosaninduced arthritis: exacerbation of joint inflammation in STAT-1 gene-knockout mice. Arthritis Rheum 2004;50:2014–23. [47] Foxwell B, Browne K, Bondeson J, Clarke C, de Martin R, Brennan F, et al. Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci U S A 1998;95:8211–5. [48] KarinM, Ben-Neriah Y.Phosphorylation meets ubiquitination: the control of NF-kappaB activity. Annu Rev Immunol 2000;18:621–63. [49] Filippin LI, Vercelino R, Marroni NP, Xavier R. Redox signalling and the inflammatory response in rheumatoid arthritis. Clin Exp Immunol 2008;152: 415–22. [50] Cardeno A, Sanchez-Hidalgo M, Aparicio-Soto M, Sanchez-Fidalgo S, Alarcon-de-la- Lastra C. Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFkB and MAPK signalling pathways. Food Funct 2014;5:1270–7. 1281M.Á. Rosillo et al. / Journal of Nutritional Biochemistry 25 (2014) 1275–1281 CAPÍTULO III Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis. (Rosillo et al. Mol Nutr Food Res. 2015. In press) Capítulo III 81 EFECTO PREVENTIVO DE UNA DIETA DE ACETATO DE HIDROXITIROSOL, UN POLIFENOL DEL ACEITE DE OLIVA VIRGEN EXTRA, EN UN MODELO DE ARTRITIS INDUCIDA POR COLÁGENO II EN RATONES Mª Ángeles Rosillo 1 , Marina Sánchez-Hidalgo 1 , Alejandro González-Benjumea 2 , José G. Fernández-Bolaños 2 , Erik Lubberts 3 , Catalina Alarcón-de-la-Lastra 1 1 Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, España 2 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, España 3 Departamento de Reumatología, Erasmus MC, Universidad Erasmus, Rotterdam, Holanda RESUMEN Antecedentes del tema: En la AR, a nivel de las articulaciones se produce hiperplasia sinovial y una infiltración masiva de células inmunes, incluyendo células T, células B, macrófagos, neutrófilos, células dendríticas, entre otras. Aunque la patogenia de la AR sigue sin conocerse en su totalidad, avances en la investigación de la enfermedad han demostrado que el aumento de los niveles de citocinas proinflamatorias como TNF-α, IL-1β, IL-6 e IFN-γ y el aumento de los niveles de metaloproteasas (MMP) en el sinovio de las articulaciones de los pacientes artríticos, juegan un papel fundamental en la erosión del cartílago articular y ósea. En la expresión génica de estos mediadores proinflamatorios, participan diversas vías de señalización celular entre las que cabe destacar las MAPKs, JAK/STAT o NF-κB, las cuales se encuentran activadas en el sinovio de pacientes con AR. La activación de estas vías de señalización, también están implicadas en la expresión de genes de proteínas proinflamatorias, como COX-2 o m-PGES-1, ambas relacionadas con el aumento de la expresión del ecosanoide proinflamatorio PGE 2. Por otro lado, el factor nuclear Nrf2, es una pieza clave en la transcripción de distintas enzimas antioxidantes como la hemo oxigenasa 1 (HO-1), se ha demostrado que la activación de la expresión génica de esta proteína es capaz de modular la respuesta inflamatoria. A pesar de los avances en la terapia farmacológica para el tratamiento de la AR, supone ciertas limitaciones, como sus efectos adversos, es por lo que en los últimos años ha crecido el interés en la utilización de suplementos dietéticos y nutracéuticos, carentes de los efectos adversos que acompaña a la farmacoterapia clásica. Estudios epidemiológicos y experimentales han puesto de manifiesto el papel beneficioso que ejercen distintos polifenoles de la dieta en la AR. AOVE, es rico en una variedad de compuestos fenólicos, entre los que se encuentra el acetato de hidroxitirosol (Ac-HT), que ha demostrado tener efectos antioxidantes y antiinflamatorios. Capítulo III 82 Objetivos: En base a los anteriores antecedentes, nos planteamos evaluar la influencia de una dieta enriquecida con Ac-HT, en un modelo de artritis experimental inducida por colágeno tipo II en ratones. Material y Métodos: Ratones DBA-1/J recién destetados, fueron distribuidos en cuatro grupos experimentales: (1) Ratones sanos alimentados con dieta estándar (CS), (2) Ratones artríticos alimentados con dita estándar (CIA), (3) Ratones artríticos alimentados con dieta enriquecida con hidroxitirosol (HT) (CIA-HT), (4) Ratones artríticos alimentados con dieta enriquecida con Ac- HT (CIA- Ac-HT). Después de seis semanas, la artritis fue inducida. El día 0, los ratones fueron inmunizados con una inyección en la base de la cola de 100 mg de colágeno II. El día 21, recibieron una inyección de refuerzo intraperitoneal, con la misma cantidad de colágeno. El grado de desarrollo de la enfermedad fue valorado de forma visual siguiendo una escala de 0-2, dónde 0=sin inflamación; 1=inflamación leve; 1.5=inflamación marcada; 2=inflamación severa y según el estudio histológico de las articulaciones. Los niveles en suero de IgG1, IgG2a, COMP y MMP-3 así como los niveles de citocinas proinflamatorias como TNF-α, IL-1β, IFN-γ, IL-6 e IL-17 en el homogenado de pata fueron determinados mediante la técnica de ELISA. Los cambios en la expresión proteica de HO-1, Nrf2 y de las proteínas implicadas en las vías de señalización celular MAPKs, JAK/STAT y NF-κB fueron estudiadas mediante western blot. Resultados: la dieta enriquecida con Ac-HT, redujo de forma significativa el edema articular y la destrucción del cartílago, previniendo el desarrollo de la AR. Los niveles séricos de las IgG1 y 2 a tanto murinas como bobinas, así como los niveles de COMP y MMP-3, los encontramos disminuidos en aquellos animales alimentados con la dieta enriquecida con Ac-HT. La expresión de las citocinas proinflamatorias TNF-α, IL-1β, IFN-γ, IL-6 e IL-17 fue menor el tejido articular de los ratones artríticos que recibieron la dieta enriquecida con el acetato. El Ac-HT produjo una inhibición en la fosforilación de la proteína STAT-3, así como de las proteínas MAPKs e inhibió la translocación al núcleo de la proteína p 65 NF-κB, previniendo la degradación de la proteína inhibitoria IκB-α. Además, Ac-HT fue capaz de aumentar la expresión proteica de Nrf2 y HO-1. Conclusión: Estos resultados ponen de manifiesto, por primera vez, el efecto antiinflamatorio de una dieta enriquecida con Ac-HT en un modelo de artritis experimental en ratones, que estuvo acompañado por una reducción de los marcadores COMP y MMP-3 en suero. Los mecanismos implicados podrían estar relacionados con una regulación de la respuesta de las células B, una activación de la vía Nrf2/HO-1 y la inhibición de la activación de distintas vías de señalización, como son JAK/STAT, MAPKs y NF- κB, implicadas en la producción de mediadores inflamatorios como citocinas y PGE 2. Concluyendo que un suplemento de AC-HT en la dieta, 6M. A. Rosillo et al. Mol. Nutr. Food Res. 2015, 0,1–10 Figure 4. (A) Expression of COX-2 and mPGES-1 in hind paws. COX-2 and mPGES-1 were determined by western blot, quantified by densitometry and normalized with respect to ␤-actin. (B) Measurement of PGE2 levels in hind paw homogenates. These were determined by ELISA kits. Data represent mean ±SEM, n= 4. **p<0.01 and ***p<0.001 versus na¨ ıve; ++p<0.01 and +++p<0.001 versus CIA; &&p<0.01 versus CIA-HTy. HTy diet showed its ineffectiveness along the experimental period. 3.9 Effects of dietary HTy-Ac on NF-␬B signaling pathway We also investigated the effects of HTy-Ac on IkB-␣degradation in cytoplasmic extracts from mice joints. As shown in Fig. 6B, I␬B-␣expression was significantly reduced in CIA-induced arthritis mice when compared with na¨ ıve mice (p<0.001 versus na¨ ıve). The I␬B-␣expression observed in CIA mice was consistent with an increase in I␬B-␣degradation thus allowing NF-␬B translocation into the nucleus to bind specific DNA sequences leading to pro-inflammatory genes transcription. According to the results obtained, the effect observed in arthritic control group on I␬B-␣protein degradation was prevented by dietary HTy-Ac treatment. On the contrary, the nuclear p65 protein levels were significantly increased in CIA group (p<0.001 versus na¨ ıve) whereas Figure 5. (A) Changes in p-STAT3 protein expression in hind paws homogenate. The expression of p-STAT3 was quantified by densitometry and normalized with respect to ␤-actin. (B) HTy-Ac dietary upregulated HO-1 and Nrf2 protein expression in hind paws homogenate. The expression was quantified by densitometry and normalized with respect to ␤-actin. Data represent mean ±SEM, n=4. ***p<0.001 versus na¨ ıve; +p<0.05 and ++p<0.01 versus CIA; &p<0.05 and &&p<0.01 versus CIA-HTy. C2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com Mol. Nutr. Food Res. 2015, 0,1–10 7 Figure 6. (A) Effect of dietary HTy-Ac on JNK, p38 and ERK (1/2) MAPKs phosphorylation in hind paws homogenate. The expression of phosphorylated proteins was expressed related to the expression of the corresponding total protein. (B) Expression of I␬B-␣in cytoplasmic fraction and p65 NF-␬B in nuclear fraction of homogenates from hind paws. The expression was quantified by densitometry and normalized with respect to ␤-actin. Data represent mean ±SEM, n=4. *p<0.05 and ***p<0.001 versus na¨ ıve; +p<0.05, ++p<0.01 versus CIA; &p<0.05 and &&p<0.01 versus CIA-HTy. dietary HTy-Ac treatment prevented the CIA-induced nuclear translocation level of p65 in paw homogenate in comparison with those arthritic animals fed with SD (p<0.01 versus CIA) (Fig. 6B) avoiding the NF-kB-mediated transcriptional activation. HTy diet failed to modify I␬B-␣degradation and nuclear p65 translocation in CIA mice. 4 Discussion Our findings, have shown, for the first time, that dietary HTy- Ac enrichment was able to prevent and down-regulate the arthritic process in the CIA model of RA. This model is commonly used to investigate relevant pathogenic mechanisms of RA as well as new antiarthritic treatments [19]. HTy-Ac was described for the first time in olive oil by Brenes et al. [20] and is found in most Spanish virgin olive oils. Moreover, recently, it was reported by Mateos et al. [21] that HTy-Ac is more soluble in the lipophilic phases than HTy, due to the presence of the ester group, which was demonstrated in a Caco-2 cell model. Thus, this increased lipophilicity suggests that HTy-Ac is better absorbed across intestinal epithelial cell monolayers than free HTy [22]. CIA induction resulted in the development of a pronounced synovitis associated with cartilage degradation and bone erosion [23]. However, dietary HTy-Ac supplementation could improve the arthritis score which was correlated with a minor migration of inflammatory cells into articular tissues in addition to a marked reduction of joint edema, synovial hyperplasia and cartilage erosion in comparison with those animals fed with SD and HTy enriched diets. A role of humoral immunity in the pathogenesis of autoimmune arthritis is suggested by evidence derived from animal models as well as patients with RA. Antibody/antigen complexes are abundantly found in the joints of RA patients and are believed to play a role in triggering the joint inflammation [24]. CIA pathogenesis is characterized by the generation of anti-CII anti-bodies. All isotypes (IgG1, IgG2a and IgG2b) of anti-CII antibodies were arthritogenic, with the IgG1 and IgG2b isotypes as the dominating arthritogenic antibodies [25]. In the present study, dietary HTy-Ac supplementation significantly decreased serum levels of IgG1-and IgG2a-anti- CII antibodies in CIA mice. These results suggest a potential role for HTy-Ac in regulating B cell responses, which may be in part, responsible for its anti-arthritic effects. The development and progression of RA is closely related to an imbalance of cytokine network. In RA synovium, elevated levels of pro-inflammatory cytokines such as TNF-␣,IL- 1␤, IL-6, IL-17 and INF-␥are produced by macrophages and synovial fibroblasts. These pro-inflammatory cytokines both directly and indirectly exert their effects through the production of additional pro-inflammatory cytokines, chemokines and MMPs at the pannus cartilage junction leading to cartilage degradation [26–28]. In addition, TNF-␣stimulates C2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com 8M. A. Rosillo et al. Mol. Nutr. Food Res. 2015, 0,1–10 osteoclastogenesis [29], suppresses the recruitment of osteoblasts and inhibit the expression of matrix genes [30], whereas IL-6 increases osteoclast numbers in trabecular bone [31]. Besides, IL-17 is a T cell-derived cytokine able to induce the release of IL-8 and IL-6, and plays a considerable role in the additive/synergistic effects induced by TNF-␣and IL-1␤ [32]. The present study showed that arthritic mice fed with HTy-Ac enriched diet had decreased IL-1␤, IL-6, IFN-␥, IL-17 and TNF-␣levels in paw homogenate in comparison with CIA mice fed with SD and HTy enriched diet. These results indicate that dietary HTy-Ac exerts anti-inflammatory activities by the blockage of IL-1␤,IL-6,IFN-␥, IL-17 and TNF-␣ production. COMP, a prominent non-collagenous component of cartilage, accounts for approximately 1% of the wet weight of articular tissue and shows great potential as a biological marker of cartilage metabolism in arthritis [33]. Increased fragments of COMP have been reported in patients with osteoarthritis, RA and joint injury, thus and the monitoring of COMP levels in synovial fluid or serum has been suggested to be a helpful method for assessing the presence and progression of arthritis. In fact, serum COMP is reduced in RA patients in remission [33]. In addition, COMP is a putative substrate for MMPs. In particular, MMP-3, is a proteinase secreted by synovial fibroblasts and chondrocytes. Its synthesis and activation is induced by various factors, including pro-inflammatory cytokines and Toll-like receptor ligand. Its activity is associated with higher joint damage in RA [34] and CIA [4] and results in degradation of aggrecan core protein, cartilage link protein, fibronectin and collagen types IV, VII, IX and XI. Likewise, serum MMP-3 level is suggested as a predictor for joint destruction in early RA or established RA and should be used in association with usual inflammatory markers to follow therapy efficiency [35]. Our data are in agreement with above studies and showed that high serum COMP and MMP-3 levels were associated with disease activity and joint progression in CIA mice, by contrast the production of both cartilage and synovial biomarkers were significantly inhibited by the HTy- Ac enriched diet in CIA mice. COX-2 and mPGES-1, enzymes responsible for the overproduction of PGE2in inflammation, are up-regulated [36] contributing to the progression of RA through EP4recep- tor activation [7]. We have shown that dietary HTy-Ac supplementation reduced PGE2 levels which could be possibly due to decreased expression of both COX-2 and mPGES-1 in the joint. Therefore, regulation of these pro-inflammatory biomarkers by HTy-Ac could represent a potential molecular target susceptible to HTy-Ac modulation, which has not been demonstrated previously. Signal transduction pathways closely involved in inflammation include the MAPKs, JAK-STAT and NF-␬B pathways [37,38]. In fact, NF-␬B nuclear transcription factor plays a pivotal role in the development and activation of Th-1 responses [39] and is responsible in addition to MAPKs for COX-2 upregulation [40]. We investigated whether the activation of NF-␬B signaling pathway was inhibited by HTy-Ac diet. Our data are in agreement with Sanchez-Fidalgo et al. that showed that HTy-Ac increased the inhibitory protein IkB-␣and reduced p65 translocation, indicating that dietary HTy-Ac inhibited the NF-␬B activation by blocking I␬B-␣degradation in dextran sulfate sodium -induced colitis in mice [16]. MAPK family members, including p38 kinases, ERKs 1 and 2 and JNKs, are involved in many important cell processes, mainly the regulation of the synthesis of chemokines, cytokines, adhesion molecules and PGs involved in RA [6,41]. Besides JNK MAPK modulates MMPs production by synovial fibroblasts and drives osteoclast differentiation in RA [42]. In particular, p38 MAPK regulates MMP-3 induction in fibroblasts [43] and osteoclast differentiation [44]. Moreover, MAPKs phosphorylate the JAK-STAT important in proinflammatory cytokine-mediated signaling pathways such as Th17 cell differentiation, leading to STAT-3 activation by phosphorylation on tyrosine residues resulting in the formation of STAT dimers that translocate into the nucleus to bind specific DNA sequences [45]. In this line, STAT-3 overexpression has been also detected in synovial membranes from RA patients and it has been reported to contribute to the chronicity of CIA induced arthritis model [4, 46]. Our findings are in concordance with above reports and showed that p38 and JNK MAPKs phosphorylation were increased in CIA control mice. Similarly, STAT-3 overexpression was also evidenced in RA synovium of control CIA mice and was positively related to the severity of synovitis, whereas dietary HTy-Ac supplementation reduced significantly both MAPKs and STAT-3 activation at transcriptional level. Collectively, our data suggest that dietary HTy-Ac may repress IL-17 production interfering negatively with JNK, p-38, p-ERK MAPKs and STAT-3 signaling pathways. Nrf2, is a redox-sensitive transcription factor and binds to antioxidant response elements (ARE) located in the promoter regions of many detoxifying/antioxidant genes, including HO-1 [47]. In inflammatory conditions, HO-1 expression protein could be part of an adaptive mechanism to limit cytotoxicity via several mechanisms including scavenging of reactive oxygen or nitrogen species, regulation of cell proliferation and prevention of apoptosis. Likewise, it has been reported that HO-1 deficiency in mice results in a chronic inflammatory state [9]. Thus, Nrf2 regulates redox status and plays key roles in cellular defense by enhancing the removal of reactive oxygen species [48]. Furthermore, it has been documented that deficiency of Nrf2 accelerates the effector phase of arthritis and aggravates joint disease [49]. In the present study, in concordance with Karatas et al. [50], expressions of Nrf2 and HO-1 were decreased in the arthritis group, by contrast dietary HTy-Ac could restore Nrf2 and HO-1 expressions conferring a role of Nrf2/HO-1 signaling in the beneficial effects of HTy-Ac in this murine model of RA. Collectively, our study has demonstrated for the first time the anti-inflammatory effects of dietary HTy-Ac in the CIA model, which were accompanied by an important reduction of the serum synovial and cartilage biomarkers COMP and C2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com Mol. Nutr. Food Res. 2015, 0,1–10 9 MMP-3. The mechanisms underlying these protective effects could be related to the regulation of B cell responses, activation of the Nrf2/HO-1 signaling and the inhibition of relevant signaling pathways such as JAK-STAT, MAPKs and NF-␬B thus controlling the production of inflammatory mediators such as Th1 cytokines and PGE2. We concluded that HTy- Ac supplement might provide a basis for developing a new dietary strategy for the prevention of RA. M.A.R. performed the experiments and data analysis. A.G.B, J.G.F.B. and E.L. performed part of the analysis. M.S.H. and C.A. designed the study and prepared the manuscript. All authors read and approved the final content of the manuscript. This study was supported by research grants AGL 2011-26949 (Ministerio de Econom´ ıa y Competitividad, ISCIII, FEDER) and P-10AGR-6609 (Junta de Andaluc´ ıa). The authors gratefully acknowledge the assistance of Center for Technology and Innovation Research, University of Seville (CITIUS). The authors have declared no conflicts of interest. 5 References [1] McInnes, I. B., Schett, G., The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 2011, 365, 2205–2219. [2] Jutley, G., Raza, K., Buckley, C. D., New pathogenic insights into rheumatoid arthritis. Curr. Opin. Rheumatol. 2015, 27, 249–255. [3] Yamanaka, H., TNF as a target of inflammation in rheumatoid arthritis. Endocr. Metab. Immune Disord. Drug Targets 2015, 15, 129–134. [4] Rosillo, M. A., Sanchez-Hidalgo, M., Sanchez-Fidalgo, S., Aparicio-Soto, M. et al., Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen-induced arthritis. Eur. J. Nutr. 2015. [5] Li, Y., Wang, S., Wang, Y., Zhou, C. et al., Inhibitory effect of the antimalarial agent artesunate on collagen-induced arthritis in rats through nuclear factor kappa B and mitogenactivated protein kinase signaling pathway. Transl. Res. 2013, 161, 89–98. [6] Criado, G., Risco, A., Alsina-Beauchamp, D., Perez-Lorenzo, M. J. et al., Alternative p38 MAPKs are essential for collageninduced arthritis. Arthritis Rheum. 2014, 66, 1208–1217. [7] McCoy, J. M., Wicks, J. R., Audoly, L. P., The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 2002, 110, 651–658. [8] Anderson, A. E., Pratt, A. G., Sedhom, M. A., Doran, J. P. et al., IL-6-driven STAT signalling in circulating CD4+lymphocytes is a marker for early anticitrullinated peptide antibodynegative rheumatoid arthritis. Ann. Rheum. Dis. 2015. [9] Minamino, T., Christou, H., Hsieh, C. M., Liu, Y. et al., Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia. Proc. Natl. Acad.Sci.USA2001, 98, 8798–8803. [10] Rosillo, M. A., Alcaraz, M. J., Sanchez-Hidalgo, M., Fernandez-Bolanos, J. G. et al., Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis. J. Nutr. Biochem. 2014, 25, 1275– 1281. [11] Mateos, R., Trujillo, M., Pereira-Caro, G., Madrona, A., et al., New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters. J. Agric. Food Chem. 2008, 56, 10960–10966. [12] Grasso, S., Siracusa, L., Spatafora, C., Renis, M., Tringali, C., Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. Bioorg. Chem. 2007, 35, 137–152. [13] Bouallagui, Z., Bouaziz, M., Lassoued, S., Engasser, J. M. et al., Hydroxytyrosol acyl esters: biosynthesis and activities. Appl. Biochem. Biotechnol. 2011, 163, 592–599. [14] Gonzalez-Correa, J. A., Navas, M. D., Lopez-Villodres, J. A., Trujillo, M. et al., Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation. Neurosci. Lett. 2008, 446, 143–146. [15] Correa, J. A., Lopez-Villodres, J. A., Asensi, R., Espartero, J. L. et al., Virgin olive oil polyphenol hydroxytyrosol acetate inhibits in vitro platelet aggregation in human whole blood: comparison with hydroxytyrosol and acetylsalicylic acid. Br. J. Nutr. 2009, 101, 1157–1164. [16] Sanchez-Fidalgo, S., Villegas, I., Aparicio-Soto, M., Cardeno, A. et al., Effects of dietary virgin olive oil polyphenols: hydroxytyrosyl acetate and 3, 4-dihydroxyphenylglycol on DSS-induced acute colitis in mice. J. Nutr. Biochem. 2015. [17] Aparicio-Soto, M., Sanchez-Fidalgo, S., Gonzalez-Benjumea, A., Maya, I. et al., Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary supplements. J. Agric. Food Chem. 2015, 63, 836–846. [18] Camporeale, A., Poli, V., IL-6, IL-17 and STAT3: a holy trinity in auto-immunity? Front. Biosci. 2012, 17, 2306–2326. [19] Ferrandiz, M. L., Maicas, N., Garcia-Arnandis, I., Terencio, M. C. et al., Treatment with a CO-releasing molecule (CORM-3) reduces joint inflammation and erosion in murine collageninduced arthritis. Ann. Rheum. Dis. 2008, 67, 1211–1217. [20] Brenes, M., Garcia, A., Garcia, P., Rios, J. J., Garrido, A., Phenolic compounds in Spanish olive oils. J. Agric. Food Chem. 1999, 47, 3535–3540. [21] Mateos, R., Pereira-Caro, G., Saha, S., Cert, R. et al., Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers. Food Chem. 2011, 125, 865–872. [22] Rubio, L., Macia, A., Valls, R. M., Pedret, A. et al., A new hydroxytyrosol metabolite identified in human plasma: hydroxytyrosol acetate sulphate. Food Chem. 2012, 134, 1132– 1136. [23] Schurgers, E., Billiau, A., Matthys, P., Collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-gamma. J. Interferon Cytokine Res. 2011, 31, 917–926. [24] Mullazehi, M., Mathsson, L., Lampa, J., Ronnelid, J., High anti-collagen type-II antibody levels and induction of proinflammatory cytokines by anti-collagen antibody-containing C2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com 10 M. A. Rosillo et al. Mol. Nutr. Food Res. 2015, 0,1–10 immune complexes in vitro characterise a distinct rheumatoid arthritis phenotype associated with acute inflammation at the time of disease onset. Ann. Rheum. Dis. 2007, 66, 537–541. [25] Nandakumar, K. S., Andren, M., Martinsson, P., Bajtner, E. et al., Induction of arthritis by single monoclonal IgG anticollagen type II antibodies and enhancement of arthritis in mice lacking inhibitory FcgammaRIIB. Eur. J. Immunol. 2003, 33, 2269–2277. [26] Komatsu, N., Takayanagi, H., Autoimmune arthritis: the interface between the immune system and joints. Adv. Immunol. 2012, 115, 45–71. [27] Noack, M., Miossec, P., Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimmun. Rev. 2014, 13, 668–677. [28] Azizi, G., Jadidi-Niaragh, F., Mirshafiey, A., Th17 Cells in Immunopathogenesis and treatment of rheumatoid arthritis. Int. J. Rheum. Dis. 2013, 16, 243–253. [29] Cenci, S., Weitzmann, M. N., Roggia, C., Namba, N. et al., Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha. J. Clin. Invest. 2000, 106, 1229– 1237. [30] Nanes, M. S., Tumor necrosis factor-alpha: molecular and cellular mechanisms in skeletal pathology. Gene 2003, 321, 1–15. [31] Jilka, R. L., Hangoc, G., Girasole, G., Passeri, G. et al., Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 1992, 257, 88–91. [32] Jeong, J. G., Kim, J. M., Cho, H., Hahn, W. et al., Effects of IL-1beta on gene expression in human rheumatoid synovial fibroblasts. Biochem. Biophys. Res. Commun. 2004, 324,3–7. [33] Saxne, T., Heinegard, D., Cartilage oligomeric matrix protein: a novel marker of cartilage turnover detectable in synovial fluid and blood. Br. J. Rheumatol. 1992, 31, 583–591. [34] Ally, M. M., Hodkinson, B., Meyer, P. W., Musenge, E. et al., Serum matrix metalloproteinase-3 in comparison with acute phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis. Mediators Inflamm. 2013, 2013, 183653. [35] Denarie, D., Constant, E., Thomas, T., Marotte, H., Could biomarkers of bone, cartilage or synovium turnover be used for relapse prediction in rheumatoid arthritis patients? Mediators Inflamm. 2014, 2014, 537324. [36] Lazarus, M., Kubata, B. K., Eguchi, N., Fujitani, Y. et al., Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages. Arch. Biochem. Biophys. 2002, 397, 336–341. [37] Morel, J., Berenbaum, F., Signal transduction pathways: new targets for treating rheumatoid arthritis. Joint Bone Spine 2004, 71, 503–510. [38] Feldmann, M., Maini, S. R., Role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics. Immunol. Rev. 2008, 223, 7–19. [39] Makarov, S. S., NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res. 2001, 3, 200–206. [40] Xie, C., Ma, L., Liu, J., Li, X. et al., SKLB023 blocks joint inflammation and cartilage destruction in arthritis models via suppression of nuclear factor-kappa B activation in macrophage. PloS One 2013, 8, e56349. [41] Li, W., Khor, T. O., Xu, C., Shen, G. et al., Activation of Nrf2- antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem. Pharmacol. 2008, 76, 1485–1489. [42] Han, Z., Boyle, D. L., Chang, L., Bennett, B. et al., c-Jun N- terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J. Clin. Invest. 2001, 108, 73–81. [43] Suzuki, M., Tetsuka, T., Yoshida, S., Watanabe, N. et al., The role of p38 mitogen-activated protein kinase in IL-6 and IL- 8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts. FEBS Lett. 2000, 465, 23–27. [44] Matsumoto, M., Sudo, T., Saito, T., Osada, H., Tsujimoto, M., Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J. Biol. Chem. 2000, 275, 31155–31161. [45] Aaronson, D. S., Horvath, C. M., A road map for those who don’t know JAK-STAT. Science 2002, 296, 1653–1655. [46] de Hooge, A. S., van de Loo, F. A., Koenders, M. I., Bennink, M. B. et al., Local activation of STAT-1 and STAT-3 in the inflamed synovium during zymosan-induced arthritis: exacerbation of joint inflammation in STAT-1 gene-knockout mice. Arthritis Rheum. 2004, 50, 2014–2023. [47] Bang, S. Y., Kim, J. H., Kim, H. Y., Lee, Y. J. et al., Achyranthes japonica exhibits anti-inflammatory effect via NF- kappaB suppression and HO-1 induction in macrophages. J. Ethnopharmacol. 2012, 144, 109–117. [48] Hayes, J. D., Dinkova-Kostova, A. T., The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem. Sci. 2014, 39, 199–218. [49] Maicas, N., Ferrandiz, M. L., Brines, R., Ibanez, L. et al., Deficiency of Nrf2 accelerates the effector phase of arthritis and aggravates joint disease. Antioxid. Redox Signal. 2011, 15, 889–901. [50] Karatas, A., Koca, S. S., Ozgen, M., Dagli, A. F. et al., Pemetrexed ameliorates experimental arthritis in rats. Inflammation 2015, 38, 9–15. C2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com RESULTADOS/DISCUSIÓN GENERAL Resultados/Discusión general 99 The focus of the chapter I has been to demonstrate the potential protective role of a diet elaborated with EVOO in the prevention and development of inflammatory arthritis and joint damage in a murine CIA-induced experimental arthritis model. This model is commonly used to investigate mechanisms relevant to RA as well as new antiarthritic treatments (Ferrandiz, Maicas et al. 2008). CIA induction resulted in the development of a pronounced synovitis associated with an autoimmune response against cartilage and production of matrix degrading enzymes accompanying cartilage degradation and bone erosions (Schurgers, Billiau et al. 2011). The results of the present work clearly indicate, for the first time, that EVOO, as the lipid component of the diet, effectively exhibited preventive and therapeutic effects in the development of inflammatory arthritis and joint damage in CIA arthritic mice in comparison with those CIA mice fed with SO (Chapter I. Figure 1 A and B). This effect was well correlated to an improved arthritis score, a minor inflammatory cells infiltration into articular tissues, reduced exudation into the synovial space, synovial hyperplasia and cartilage erosion (Chapter I. Figure 2). The pathogenesis of RA is closely related to an imbalance of cytokine network contributing to the development and progression of both CIA and RA (Komatsu and Takayanagi 2012). Actually, overexpression of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-17 may activate osteoclasts and macrophages and also recruit leukocytes in inflamed joints. The infiltrated leukocytes produce cytokines, chemokines and tissue destructive enzymes leading to chronic inflammation, cartilage damage and bone erosion. Besides, IL-17 is able to induce the release of IL-8, IL-6 and plays a remarkable role in the additive/synergistic effects induced by TNF-α and IL-1β (Jeong, Kim et al. 2004). Importantly, targeting IL-17 has demonstrated therapeutic effects in animal models of inflammatory and autoimmune diseases, including RA (Miossec and Kolls 2012). Cytokines also up-regulate MMPs expression by chondrocytes and synoviocytes at the pannus cartilage junction leading to cartilage degradation (Fossiez, Djossou et al. 1996, Azizi, Jadidi-Niaragh et al. 2013, Noack and Miossec 2014). Therefore, we examined TNF-α, IL-1ß and IL-17 levels in paw homogenates from CIA animals in comparison with naïve control group. Our results indicate that animals fed with EVOO diet showed a significant reduction in all pro inflammatory cytokines levels. Consequently, EVOO intake would contribute to reduce the inflammatory response and joint damage by suppressing the production of the key Th17 polarisation cytokines, TNF- α, and IL-1β, in CIA arthritic mice (Chapter I. Figure 4). COMP, is a matrix protein with a great potential as a biological marker of cartilage metabolism in arthritis (Saxne and Heinegard 1992). Increased fragments of COMP have been reported in patients with osteoarthritis, RA and joint injury; thus, the monitoring of COMP levels in synovial Resultados/Discusión general 100 fluid or serum has been suggested to be a helpful method for assessing the presence and progression of the inflammatory disease. In this sense, serum COMP levels are reduced in RA patients in remission (Saxne and Heinegard 1992, Lai, Yu et al. 2012). In addition, COMP is a putative substrate for MMPs. Particularly, MMP-3, is a proteinase secreted by synovial fibroblasts and chondrocytes. Its activity results in degradation of aggrecan core protein, cartilage link protein, fibronectin, and collagen types IV, VII, IX, and XI. MMP-3 is present in RA synovial fluid and overexpressed in rheumatoid synovium and then, associated with higher joint damage in RA (Ally, Hodkinson et al. 2013). Likewise, serum MMP-3 level is suggested as a predictor for joint destruction in early RA or established RA and should be used in association with usual inflammatory markers to follow therapy efficiency (Denarie, Constant et al. 2014). Our data are in agreement with above studies and showed that high serum COMP and MMP-3 levels were associated with disease activity and joint progression in CIA mice, by contrast the production of both cartilage and synovial biomarkers were significantly inhibited by dietary EVOO treatment in CIA mice (Chapter I. Figure 3). Abnormal signalling pathways play an important role in the inflammatory process and can lead to dysregulation of the inflammatory response being crucial in RA pathogenesis. Signal transduction pathways closely involved in inflammation included the MAPKs, JAK-STAT and NF- κB pathways (Morel and Berenbaum 2004). NF-κB is a crucial transcriptional activator for the expression of multiple proinflammatory genes in the microenvironment of the arthritic joints, including TNF-α, IL-1β, IL-6 and IL-17, that exert their influences on both osteoclasts and osteoblasts differentiation and thereby contributing to progressive joint destruction in RA patients and CIA mice (Okamoto, Yoshio et al. 2010, Molinero, Cubre et al. 2012) as mentioned above. In fact, several anti-rheumatic drugs, currently in clinical use, inhibit NF-κB activation (Hwang, Noh et al. 2013). Interestingly, intra-articular or systemic blockade of NF-κB signalling is an effective target in the treatment of arthritis in animal models of RA (Min, Yan et al. 2015). Taken together, these studies confirm the importance of NF-κB activation in the development of RA. Consequently, NF-kB pathway suppression could be a novel strategy for delaying the progress of RA. Our results suggested that dietary EVOO treatment suppressed NF-κB activation (Chapter I. Figure 8), down-regulating pro-inflammatory cytokines and MMP-3 expression thus minimizing joint destruction in CIA-induced arthritic mice. MAPK family members, including p38 kinases, ERKs 1 and 2, and JNKs, play critical roles in many important cell processes, including cell division, differentiation, inflammation, and apoptosis. They are especially drawing attention in RA because of the involvement in regulating both cytokine production and cytokine action (Criado, Risco et al. 2014, Li, Li et al. 2014). In fact, JNK MAPK regulates MMPs production by Resultados/Discusión general 101 synovial fibroblasts and drives osteoclast differentiation in RA (Han, Boyle et al. 2001) and p38 MAPK regulates MMP-3 induction in fibroblasts (Suzuki, Tetsuka et al. 2000) and osteoclast differentiation (Matsumoto, Sudo et al. 2000). Moreover, MAPKs phosphorylate the janus kinase signal transducer and activator of transcription (JAK-STAT) important in proinflammatory cytokine-mediated signalling pathway leading to STAT-3 activation (Aaronson and Horvath 2002). STAT-3 has been described as a critical transcription factor involved in Th17 cell differentiation (Park, Lim et al. 2013). In this line, STAT-3 overexpression has been also detected in synovial membranes from RA patients and it has been reported to contribute to the chronicity of inflammation in a murine zymosan-induced arthritis model (de Hooge, van de Loo et al. 2004). Recently, STA-21, a promising STAT-3 inhibitor has shown antiarthritic effects in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice, an animal model of RA (Park, Kwok et al. 2014). Our study showed that p38 and JNK MAPKs phosphorylations were increased in SO-CIA mice. Similarly, STAT-3 overexpression was also evidenced in RA synovium of SO-CIA mice and was positively related to the severity of synovitis. Whereas EVOO diet intake reduced significantly both MAPKs and STAT-3 activation at transcriptional level (Chapter I. Figures 5 and 7). Altogether, our results suggest that dietary EVOO may repress IL-17 production interfering negatively with JNK and p-38 MAPKs and STAT-3 signalling pathways. HO-1 activity catabolises heme to carbon monoxide, iron and biliverdin, the latter being reduced to bilirubin by biliverdin reductase. Nrf2 plays a central role for expression of HO-1. In basal conditions, Nrf2 is sequestered in the cytoplasm by Kelch like ECH-associated protein1 (Keap1) and degraded by the ubiquitin dependent 26S proteasome system. Under activation, Nrf2 released from Keap1 inhibition, translocate to the nucleus, heterodimerizes with Maf, and binds antioxidant response elements (AREs) located in the promoter regions of many detoxifying/antioxidant genes, including HO-1 (Bang, Kim et al. 2012). In inflammatory and immune conditions the expression of this protein could be part of an adaptative mechanism to limit cytotoxicity via several mechanisms including scavenging of reactive oxygen or nitrogen species, regulation of cell proliferation and prevention of apoptosis (Ferrandiz, Maicas et al. 2008). Likewise, it has been reported that HO-1 deficiency in mice results in a chronic inflammatory state (Minamino, Christou et al. 2001). Our data showed that HO-1 could represent a potential molecular target susceptible to EVOO modulation, which has not been demonstrated previously, since dietary EVOO treatment strongly augmented Nrf2 and HO-1 expression conferring a role of HO-1 in the beneficial effects of EVOO in this murine model of chronic inflammation (Chapter I. Figure 6). Since our results show that EVOO diet was more effective in reducing arthritis severity in comparison with SO diet, we could suggest that the responsibility for such beneficial Resultados/Discusión general 102 properties could to be assigned to both an adequate fatty acid profile of EVOO and the presence of a high proportion of phenolic compounds. In this sense, a recent report showed that oleuropein aglycone, a polyphenol found in olive oil, exerted an anti-inflammatory effect and ameliorated the joint damage associated with CIA (Impellizzeri, Esposito et al. 2011). In addition, these improved effects observed could be due to a possible synergistic effect among EVOO constituents, since it is not clear whether all the possible beneficial mechanisms act independently of each other or whether they have a synergistic or competitive action (Lopez- Miranda, Perez-Jimenez et al. 2010, de la Lastra Romero 2011). In the chapter II, we elucidated the effects of oral PE from EVOO treatment in murine CIA and study, the biochemical routes and signalling pathway involved. According to our results, we have demonstrated that oral administration of PE is able to down-regulate the arthritic process in the CIA model of RA (Chapter II. Figure 1 A and B). PE showed anti-inflammatory effects with reductions in joint oedema and migration of inflammatory cells. In addition, PE protected joints against cartilage alterations and bone erosion (Chapter II. Figure2). The progression of arthritis is associated with sustained production of proinflammatory cytokines (Goronzy and Weyand 2009). We have shown in the CIA model that PE controls the local levels of these cytokines driving the production of different inflammatory and catabolic mediators (Chapter II. Figure 3). These findings are in agreement with ours previous results evaluated in CIA mice fed with dietary EVOO. Particularly, proinflammatory cytokines induce further cytokines, chemokines, eicosanoids and reactive oxygen species amplifying the inflammatory response (Abramson and Amin 2002). Therefore, COX-2 and mPGES-1, enzymes responsible for the overproduction of PGE2 in inflammation, are up-regulated (Lazarus, Kubata et al. 2002) contributing to the progression of RA through EP4 receptor activation (McCoy, Wicks et al. 2002). We have shown that PE reduces PGE 2 levels (Chapter II. Figure 3), which would be dependent on the downregulation of COX-2 and mPGES-1 expression in the joint (Chapter II. Figure 4 A and B). Cytokines also induce the expression of matrix metalloproteinases at the pannus cartilage junction leading to cartilage degradation (Okada, Nagase et al. 1987, Ribbens, Martin y Porras et al. 2002). In addition, TNFα stimulates osteoclastogenesis (Cenci, Weitzmann et al. 2000), suppresses the recruitment of osteoblasts and inhibits the expression of matrix genes (Nanes 2003) whereas IL- 6 increases osteoclast numbers in trabecular bone (Jilka, Hangoc et al. 1992). Therefore, inhibition of proinflammatory cytokine production by PE would result in the reduction of the inflammatory response and tissue damage. MAPK regulate the synthesis of chemokines, cytokines, adhesion molecules and PGs involved in inflammation. In addition, they mediate the Resumen/ Abstract 114 many of the beneficial effects of EVOO intake are due to its minor highly bioactive components (about 1–2 % of oil weight) (Alarcon de la Lastra et al. 2001) Among them, phenolic compounds such as hydroxytyrosol, tyrosol and oleuropein have shown anti-inflammatory and antioxidant effects (Omar et al. 2010). Current experimental studies support a beneficial role of polyphenols from EVOO in several inflammatory diseases, including RA (Martinez-Dominguez, de la Puerta, and Ruiz-Gutierrez 2001; Impellizzeri et al. 2011; Gong et al. 2009). Although EVOO has demonstrated anti-inflammatory effects, it has not reported so much evidence of its possible immunomodulatory effects. Therefore, the objectives of this thesis were: 1. To determinate the possible protective effect of dietary extra virgin olive oil (EVOO) in collagen-induced arthritis (CIA) in DBA 1/J mice, an experimental model of RA and explore the biochemical routes and possibly intracellular signalling pathways. 2. To evaluate the oral polyphenolic extract from EVOO treatment in murine collageninduced arthritis and study the biochemical routes and signalling pathway involved. 3. To investigate the effects of hydroxytyrosol (HTy) or hydroxytyrosol acetate (HTy- Ac), polyphenolics compounds from EVOO enriched-diets in experimental arthritis model in mice and elucidate the molecular mechanisms and signalling pathways involved. Results and Discussion 1. Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen‑induced arthritis. (Rosillo et al. Eur J Nutr. 2015 In press) Three-week-old male DBA-1/J mice were randomized into four experimental groups: (1) Sham sunflower diet (SO-Sham) group received a diet elaborated with a marketable sunflower oil; (2) CIA sunflower diet (SO-CIA) group received a diet elaborated with a marketable sunflower oil; (3) Sham EVOO diet (EVOO-Sham) group were fed with a diet made with a marketable EVOO picual variety and (4) CIA EVOO diet (EVOO-CIA) group were fed with a diet made with a marketable EVOO picual variety. After 6 weeks, arthritis was induced by type II collagen. Resumen/Abstract 115 Experiments followed a protocol approved by the Animal Ethics Committee of the University of Seville, and all experiments were in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Counsel 2010/630/EU) Our results revealed, that EVOO, as the lipid component of the diet, effectively exhibited preventive and therapeutic effects in the development of inflammatory arthritis and joint damage in CIA arthritic mice in comparison with those CIA mice fed with SO. This effect was correlated to an improved arthritis score, a minor inflammatory cells infiltration into articular tissues, reduced exudation into the synovial space, synovial hyperplasia and cartilage erosion. Overexpression of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-17 may activate osteoclasts and macrophages and recruit leukocytes in inflamed joints. Besides, it is well-known that IL-17 is able to induce the release of IL-8 and IL-6, and plays a remarkable role in the additive/synergistic effects induced by TNF-α and IL-1β (Jeong et al. 2004). Our results indicate that animals fed with EVOO diet showed a significant reduction in IL-1β, TNF-α and IL-17 proinflammatory cytokines levels in paw homogenates. Cartilage oligomeric matrix protein (COMP) is a matrix protein with a great potential as a biological marker of cartilage metabolism in arthritis (Saxne and Heinegard 1992). In addition, COMP is a putative substrate for metalloproteinases (MMPs). Particularly, MMP-3 is a proteinase secreted by synovial fibroblasts and chondrocytes and its activity results in degradation of aggrecan core protein, cartilage link protein, fibronectin and collagen. Our data showed that the production of both cartilage (COMP) and synovial (MMP-3) biomarkers was significantly inhibited by dietary EVOO treatment in CIA mice. Abnormal signalling pathways play an important role in the inflammatory process and can lead to a dysregulation of the inflammatory response being crucial in RA pathogenesis. Nuclear factor κB (NF-κB) is a crucial transcriptional activator for the expression of multiple proinflammatory genes involved in the microenvironment of the arthritic joints, playing an important role in the development of RA. (Morel and Berenbaum 2004; Okamoto et al. 2010). Our results suggested that dietary EVOO treatment suppressed NF-κB activation in CIA-induced arthritic mice. Similarly, the mitogen-activated protein kinase (MAPK) family also plays critical roles in RA pathogenesis (Han et al. 2001; Suzuki et al. 2000) regulating cytokine production, and activating the janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathway through STAT-3 phosphorylation (Aaronson and Horvath 2002). Our results demonstrated that EVOO diet intake reduced significantly both MAPKs and STAT-3 activation. On the other hand, nuclear factor E2-related factor 2 (Nrf2) plays a central role for expression Resumen/ Abstract 116 of heme oxigenase 1 (HO-1), antioxidant enzyme. Our data showed that HO-1 could represent a potential molecular target susceptible to EVOO modulation, since dietary EVOO treatment strongly augmented Nrf2 and HO-1 protein expression conferring a role of HO-1 in the beneficial effects of EVOO in this murine model of chronic inflammation. Altogether, our results confirm, for the first time, that EVOO intake dramatically attenuated the progression and severity of arthritis in CIA DBA/1 J mice through Nrf2/HO-1 upregulation and NF-κB, MAPKs and JAK-STAT signalling pathway inhibition, decreasing the inflammatory cascade induced by CIA. 2. Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis. (Rosillo et al. Pharmacol Res. 2012;66(3):235-42.) CIA was induced in nine-weeks-old male DBA-1/J mice. On day 29, animals were randomized in four groups of mice: naïve group (NA), CIA group (CIA) and two treatment groups: CIA mice with polyphenolic extract (PE) treatment (PE 100 and 200 mg/kg, orally, once a day from days 29 to 41). In this study, we have demonstrated that oral administration of PE was able to downregulate the arthritic process in the CIA model of RA. PE showed anti-inflammatory effects with reductions in joint edema and migration of inflammatory cells. In addition, PE protected joints against cartilage alterations and bone erosion. The progression of arthritis is associated with sustained production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 (Goronzy and Weyand 2009). Our data shown that PE treatment controls the local levels of these cytokines, driving the production of different inflammatory and catabolic mediators in CIA model. In this sense, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1), enzymes responsible for the overproduction of PGE 2 in inflammation, are dramatically up-regulated (Lazarus et al. 2002) contributing to the progression of RA through EP 4 receptor activation (McCoy, Wicks, and Audoly 2002). Our data suggest that PE reduces PGE 2 levels, which would be dependent on the down-regulation of COX-2 and mPGES-1 protein expression in the joint. In addition, PE treatment inhibits the phosphorilation of JNK and p38 MAPK and transcription factor STAT-3. Similarly, PE addition decreases NF-κB nuclear translocation leading to the down-regulation of the arthritic process. Resumen/Abstract 117 In summary, our study has demonstrated, for the first time, the anti-inflammatory and joint protective effects of PE from EVOO in a CIA-induced experimental model, which would be related to the inhibition of relevant signalling pathways such as NF-κB, JAK/STAT and MAPKs controlling the production of inflammatory mediators. 3. Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis. (Rosillo et al. Mol Nutr Food Res. 2015. In press) Three-weeks-old male DBA-1/J mice, were randomized into four experimental groups during all experimental period: (i) naïve group received a standard diet (SD), (ii) CIA group received a SD, (iii) CIA-HTy group received a 0.05% HTy diet and (iv) CIA-HTy-Ac group received a 0.05% HTy-Ac diet. After 6 weeks, arthritis was induced by type II collagen. HTy-Ac was described for the first time in olive oil by Brenes et al. (Brenes et al. 1999) and is found in most Spanish virgin olive oils. Moreover, recently, it was reported that HTy-Ac is more soluble in the lipophilic phases than HTy, due to the presence of the ester group. Thus, this increased lipophilicity suggests that HTy-Ac is better absorbed across intestinal epithelial cell monolayers than free HTy (Rubio et al. 2012). Our findings have revealed that dietary HTy-Ac supplementation could improve the arthritis score which was correlated with a minor migration of inflammatory cells into articular tissues in addition to a marked reduction of joint edema, synovial hyperplasia and cartilage erosion in comparison with those animals fed with standard diet (SD) and hydroxytyrosol (HTy) enriched diets. CIA pathogenesis is characterized by the generation of anti-CII anti-bodies (Nandakumar et al. 2003). All isotypes (IgG1, IgG2a and IgG2b) of anti-CII antibodies were arthritogenic, with the IgG1 and IgG2b isotypes as the dominating arthritogenic antibodies (Nandakumar et al. 2003). Our results demonstrated that dietary HTy-Ac supplementation significantly decreased serum levels of IgG1-and IgG2a-anti-CII antibodies in CIA mice. These results suggest a potential role for HTy-Ac in regulating B cell responses, which may be in part, responsible for its antiarthritic effects. Our results shown that arthritic mice fed with HTy-Ac enriched diet had decreased serum COMP and MMP-3 levels, as well as, pro-inflammatory cytokines such as IL-1β, IL-6, IFN- γ, IL-17 and TNF-α levels in paw homogenate in comparison with CIA mice fed with SD and HTy Resumen/ Abstract 118 enriched diet. Moreover, the activation of JAK/STAT, MAPKs and NF-κB pathways were drastically ameliorated whereas Nrf2 and HO-1 protein expressions were significantly upregulated in those mice fed with HTy-Ac. As a conclusion, our study reveals, for the first time, the anti-inflammatory effects of dietary HTy-Ac in the CIA model. The mechanisms underlying these protective effects could be related to the regulation of B cell responses, activation of the Nrf2/HO-1 signalling pathway and the inhibition of relevant signalling pathways such as JAK-STAT, MAPKs and NF-κB controlling the production of inflammatory mediators. Conclusion Altogether, our results suggest that EVOO exerts preventive effects in the development of experimental RA, playing its multiple minor components a key role in these healthy benefits. Thus, EVOO may be considered as supportive nutritional therapy for RA patients as well as its poliphenolic fraction might provide an attractive nutraceutical complement in management of RA, mainly when the effects of inexpensive, side effect-free therapies based on dietary EVOO might suppose an improvement in public health on the prevention of chronic pathologies with high prevalence in the population. BIBLIOGRAFÍA Bibliografía 121 Aaronson, D. S. and C. M. Horvath (2002). "A road map for those who don't know JAK-STAT." Science 296(5573): 1653-1655. Abendroth, A., A. Michalsen, R. Ludtke, A. Ruffer, F. Musial, G. J. Dobos and J. Langhorst (2010). "Changes of Intestinal Microflora in Patients with Rheumatoid Arthritis during Fasting or a Mediterranean Diet." Forsch Komplementmed 17(6): 307-313. Abramson, S. B. and A. Amin (2002). "Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage." Rheumatology (Oxford) 41(9): 972-980. Ahmed, S., H. Marotte, K. Kwan, J. H. Ruth, P. L. Campbell, B. J. Rabquer, A. Pakozdi and A. E. Koch (2008). "Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production." Proc Natl Acad Sci U S A 105(38): 14692-14697. Ahmed, S., A. Pakozdi and A. E. Koch (2006). "Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts." Arthritis Rheum 54(8): 2393-2401. Ahmed, S., M. D. Silverman, H. Marotte, K. Kwan, N. Matuszczak and A. E. Koch (2009). "Downregulation of myeloid cell leukemia 1 by epigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor alpha-induced apoptosis." Arthritis Rheum 60(5): 1282-1293. Alarcon de la Lastra, C., M. D. Barranco, M. J. Martin, J. Herrerias and V. Motilva (2002). "Extravirgin olive oil-enriched diets reduce indomethacin-induced gastric oxidative damage in rats." Dig Dis Sci 47(12): 2783-2790. Alarcon de la Lastra, C., M. D. Barranco, V. Motilva and J. M. Herrerias (2001). "Mediterranean diet and health: biological importance of olive oil." Curr Pharm Des 7(10): 933-950. Ally, M. M., B. Hodkinson, P. W. Meyer, E. Musenge, M. Tikly and R. Anderson (2013). "Serum matrix metalloproteinase-3 in comparison with acute phase proteins as a marker of disease activity and radiographic damage in early rheumatoid arthritis." Mediators Inflamm 2013: 183653. Anderson, A. E., A. G. Pratt, M. A. Sedhom, J. P. Doran, C. Routledge, B. Hargreaves, P. M. Brown, K. A. Le Cao, J. D. Isaacs and R. Thomas (2015). "IL-6-driven STAT signalling in circulating CD4+ lymphocytes is a marker for early anticitrullinated peptide antibody-negative rheumatoid arthritis." Ann Rheum Dis. Andersson, A. K., C. Li and F. M. Brennan (2008). "Recent developments in the immunobiology of rheumatoid arthritis." Arthritis Res Ther 10(2): 204. Aparicio-Soto, M., S. Sanchez-Fidalgo, A. Gonzalez-Benjumea, I. Maya, J. G. Fernandez-Bolanos and C. Alarcon-de-la-Lastra (2015). "Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary supplements." J Agric Food Chem 63(3): 836-846. Aparicio-Soto, M., M. Sanchez-Hidalgo, A. Cardeno, M. A. Rosillo, S. Sanchez-Fidalgo, J. Utrilla, I. Martin-Lacave and C. Alarcon-de-la-Lastra (2015). "Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf-2 antioxidant pathway and suppression of JAK/STAT, NF-kappaB and MAPK activation." J Nutr Biochem. Bibliografía 122 Arora, R., A. Kuhad, I. P. Kaur and K. Chopra (2015). "Curcumin loaded solid lipid nanoparticles ameliorate adjuvant-induced arthritis in rats." Eur J Pain 19(7): 940-952. Aupperle, K., B. Bennett, Z. Han, D. Boyle, A. Manning and G. Firestein (2001). "NF-kappa B regulation by I kappa B kinase-2 in rheumatoid arthritis synoviocytes." J Immunol 166(4): 2705- 2711. Azizi, G., F. Jadidi-Niaragh and A. Mirshafiey (2013). "Th17 Cells in Immunopathogenesis and treatment of rheumatoid arthritis." Int J Rheum Dis 16(3): 243-253. Bang, S. Y., J. H. Kim, H. Y. Kim, Y. J. Lee, S. Y. Park, S. J. Lee and Y. Kim (2012). "Achyranthes japonica exhibits anti-inflammatory effect via NF-kappaB suppression and HO-1 induction in macrophages." J Ethnopharmacol 144(1): 109-117. Berbert, A. A., C. R. Kondo, C. L. Almendra, T. Matsuo and I. Dichi (2005). "Supplementation of fish oil and olive oil in patients with rheumatoid arthritis." Nutrition 21(2): 131-136. Bermudez, B., S. Lopez, A. Ortega, L. M. Varela, Y. M. Pacheco, R. Abia and F. J. Muriana (2011). "Oleic acid in olive oil: from a metabolic framework toward a clinical perspective." Curr Pharm Des 17(8): 831-843. Bouallagui, Z., M. Bouaziz, S. Lassoued, J. M. Engasser, M. Ghoul and S. Sayadi (2011). "Hydroxytyrosol acyl esters: biosynthesis and activities." Appl Biochem Biotechnol 163(5): 592- 599. Bradford, M. M. (1976). "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding." Anal Biochem 72: 248-254. Brenes, M., A. Garcia, P. Garcia, J. J. Rios and A. Garrido (1999). "Phenolic compounds in Spanish olive oils." J Agric Food Chem 47(9): 3535-3540. Brennan, F. M. and I. B. McInnes (2008). "Evidence that cytokines play a role in rheumatoid arthritis." J Clin Invest 118(11): 3537-3545. Brooks, P. M. (2006). "The burden of musculoskeletal disease--a global perspective." Clin Rheumatol 25(6): 778-781. Bruneton, J. (2001). Compuestos Fenólicos. Farmacognosia. Zaragoza, Ed. Acribia S.A.: 227-454. Brzustewicz, E. and E. Bryl (2015). "The role of cytokines in the pathogenesis of rheumatoid arthritis - Practical and potential application of cytokines as biomarkers and targets of personalized therapy." Cytokine 76(2): 527-536. Buhrmann, C., A. Mobasheri, U. Matis and M. Shakibaei (2010). "Curcumin mediated suppression of nuclear factor-kappaB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment." Arthritis Res Ther 12(4): R127. Byun, H. S., J. K. Song, Y. R. Kim, L. Piao, M. Won, K. A. Park, B. L. Choi, H. Lee, J. H. Hong, J. Park, J. H. Seok, Y. J. Lee, S. W. Kang and G. M. Hur (2008). "Caspase-8 has an essential role in resveratrol-induced apoptosis of rheumatoid fibroblast-like synoviocytes." Rheumatology (Oxford) 47(3): 301-308. Calder, P. C. (2011). "Fatty acids and inflammation: the cutting edge between food and pharma." Eur J Pharmacol 668 Suppl 1: S50-58. Calder, P. C. (2012). "The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability." Mol Nutr Food Res 56(7): 1073-1080. Bibliografía 123 Calder, P. C. (2015). "Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance." Biochim Biophys Acta 1851(4): 469-484. Camporeale, A. and V. Poli (2012). "IL-6, IL-17 and STAT3: a holy trinity in auto-immunity?" Front Biosci (Landmark Ed) 17: 2306-2326. Canter, P. H., B. Wider and E. Ernst (2007). "The antioxidant vitamins A, C, E and selenium in the treatment of arthritis: a systematic review of randomized clinical trials." Rheumatology (Oxford) 46(8): 1223-1233. Cardeno, A., M. K. Magnusson, H. Strid, C. Alarcon de La Lastra, M. Sanchez-Hidalgo and L. Ohman (2014). "The unsaponifiable fraction of extra virgin olive oil promotes apoptosis and attenuates activation and homing properties of T cells from patients with inflammatory bowel disease." Food Chem 161: 353-360. Cardeno, A., M. Sanchez-Hidalgo and C. Alarcon-de-la-Lastra (2013). "An up-date of olive oil phenols in inflammation and cancer: molecular mechanisms and clinical implications." Curr Med Chem 20(37): 4758-4776. Cardeno, A., M. Sanchez-Hidalgo, M. Aparicio-Soto, S. Sanchez-Fidalgo and C. Alarcon-de-la- Lastra (2014). "Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFkappaB and MAPK signalling pathways." Food Funct 5(6): 1270-1277. Carlin, G., R. Djursater and G. Smedegard (1992). "Sulphasalazine inhibition of human granulocyte activation by inhibition of second messenger compounds." Ann Rheum Dis 51(11): 1230-1236. Cascao, R., H. S. Rosario, M. M. Souto-Carneiro and J. E. Fonseca (2010). "Neutrophils in rheumatoid arthritis: More than simple final effectors." Autoimmun Rev 9(8): 531-535. Cenci, S., M. N. Weitzmann, C. Roggia, N. Namba, D. Novack, J. Woodring and R. Pacifici (2000). "Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha." J Clin Invest 106(10): 1229-1237. Cohen, S., E. Hurd, J. Cush, M. Schiff, M. E. Weinblatt, L. W. Moreland, J. Kremer, M. B. Bear, W. J. Rich and D. McCabe (2002). "Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial." Arthritis Rheum 46(3): 614-624. Cohen, S. B., P. Emery, M. W. Greenwald, M. Dougados, R. A. Furie, M. C. Genovese, E. C. Keystone, J. E. Loveless, G. R. Burmester, M. W. Cravets, E. W. Hessey, T. Shaw, M. C. Totoritis and R. T. Group (2006). "Rituximab for rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter, randomized, double-blind, placebo-controlled, phase III trial evaluating primary efficacy and safety at twenty-four weeks." Arthritis Rheum 54(9): 2793- 2806. Corps, A. N., V. A. Curry, D. J. Buttle, B. L. Hazleman and G. P. Riley (2004). "Inhibition of interleukin-1beta-stimulated collagenase and stromelysin expression in human tendon fibroblasts by epigallocatechin gallate ester." Matrix Biol 23(3): 163-169. Correa, J. A., J. A. Lopez-Villodres, R. Asensi, J. L. Espartero, G. Rodriguez-Gutierez and J. P. De La Cruz (2009). "Virgin olive oil polyphenol hydroxytyrosol acetate inhibits in vitro platelet aggregation in human whole blood: comparison with hydroxytyrosol and acetylsalicylic acid." Br J Nutr 101(8): 1157-1164. Bibliografía 124 Creemers MCW, V. d. P. L. (2004). Rheumatoid arthritis – the clinical picture. OXFORD TEXTBOOK OF RHEUMATOLOGY. M. P. Isenberg DA, Woo P, Klars D, Breedveld FC Oxford, Oxford University Press. Criado, G., A. Risco, D. Alsina-Beauchamp, M. J. Perez-Lorenzo, A. Escos and A. Cuenda (2014). "Alternative p38 MAPKs are essential for collagen-induced arthritis." Arthritis Rheumatol 66(5): 1208-1217. Cronstein, B. N. (2005). "Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis." Pharmacol Rev 57(2): 163-172. Cronstein, B. N. (2007). "Interleukin-6--a key mediator of systemic and local symptoms in rheumatoid arthritis." Bull NYU Hosp Jt Dis 65 Suppl 1: S11-15. Cutolo, M. (2007). "Sex and rheumatoid arthritis: mouse model versus human disease." Arthritis Rheum 56(1): 1-3. Cuzzocrea, S., E. Mazzon, L. Dugo, N. S. Patel, I. Serraino, R. Di Paola, T. Genovese, D. Britti, M. De Maio, A. P. Caputi and C. Thiemermann (2003). "Reduction in the evolution of murine type II collagen-induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma." Arthritis Rheum 48(12): 3544-3556. Chang, K., S. M. Yang, S. H. Kim, K. H. Han, S. J. Park and J. I. Shin (2014). "Smoking and rheumatoid arthritis." Int J Mol Sci 15(12): 22279-22295. Chapuy-Regaud, S., L. Nogueira, C. Clavel, M. Sebbag, C. Vincent and G. Serre (2005). "IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin." Clin Exp Immunol 139(3): 542-550. Chauhan, P. S., N. K. Satti, P. Sharma, V. K. Sharma, K. A. Suri and S. Bani (2012). "Differential effects of chlorogenic acid on various immunological parameters relevant to rheumatoid arthritis." Phytother Res 26(8): 1156-1165. Chen, X., J. Lu, M. An, Z. Ma, H. Zong and J. Yang (2014). "Anti-inflammatory effect of resveratrol on adjuvant arthritis rats with abnormal immunological function via the reduction of cyclooxygenase-2 and prostaglandin E2." Mol Med Rep 9(6): 2592-2598. Chiba, T., K. Sato, M. Endo, T. Ando, M. Inomata, S. Orii and K. Suzuki (2005). "Upper gastrointestinal disorders induced by non-steroidal anti-inflammatory drugs." Hepatogastroenterology 52(64): 1134-1138. Chimenti, M. S., P. Triggianese, P. Conigliaro, E. Candi, G. Melino and R. Perricone (2015). "The interplay between inflammation and metabolism in rheumatoid arthritis." Cell Death Dis 6: e1887. Cho, M. L., C. H. Yoon, S. Y. Hwang, M. K. Park, S. Y. Min, S. H. Lee, S. H. Park and H. Y. Kim (2004). "Effector function of type II collagen-stimulated T cells from rheumatoid arthritis patients: crosstalk between T cells and synovial fibroblasts." Arthritis Rheum 50(3): 776-784. Choi, E. M. and Y. S. Lee (2010). "Luteolin suppresses IL-1beta-induced cytokines and MMPs production via p38 MAPK, JNK, NF-kappaB and AP-1 activation in human synovial sarcoma cell line, SW982." Food Chem Toxicol 48(10): 2607-2611. De Bosscher, K., M. L. Schmitz, W. Vanden Berghe, S. Plaisance, W. Fiers and G. Haegeman (1997). "Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation." Proc Natl Acad Sci U S A 94(25): 13504-13509. Bibliografía 131 Manoury-Schwartz, B., G. Chiocchia, N. Bessis, O. Abehsira-Amar, F. Batteux, S. Muller, S. Huang, M. C. Boissier and C. Fournier (1997). "High susceptibility to collagen-induced arthritis in mice lacking IFN-gamma receptors." J Immunol 158(11): 5501-5506. Marder, W., S. Khalatbari, J. D. Myles, R. Hench, S. Lustig, S. Yalavarthi, A. Parameswaran, R. D. Brook and M. J. Kaplan (2013). "The peroxisome proliferator activated receptor-gamma pioglitazone improves vascular function and decreases disease activity in patients with rheumatoid arthritis." J Am Heart Assoc 2(6): e000441. Martinez-Dominguez, E., R. de la Puerta and V. Ruiz-Gutierrez (2001). "Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet." Inflamm Res 50(2): 102-106. Mateos, R., M. Trujillo, G. Pereira-Caro, A. Madrona, A. Cert and J. L. Espartero (2008). "New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters." J Agric Food Chem 56(22): 10960-10966. Matsumoto, M., T. Sudo, T. Saito, H. Osada and M. Tsujimoto (2000). "Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL)." J Biol Chem 275(40): 31155-31161. McCoy, J. M., J. R. Wicks and L. P. Audoly (2002). "The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis." J Clin Invest 110(5): 651-658. McInnes, I. B. and G. Schett (2011). "The pathogenesis of rheumatoid arthritis." N Engl J Med 365(23): 2205-2219. Medina, E., A. de Castro, C. Romero and M. Brenes (2006). "Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity." J Agric Food Chem 54(14): 4954-4961. Messemaker, T. C., T. W. Huizinga and F. Kurreeman (2015). "Immunogenetics of rheumatoid arthritis: Understanding functional implications." J Autoimmun 64: 74-81. Miles, E. A. and P. C. Calder (2012). "Influence of marine n-3 polyunsaturated fatty acids on immune function and a systematic review of their effects on clinical outcomes in rheumatoid arthritis." Br J Nutr 107 Suppl 2: S171-184. Mima, T. and N. Nishimoto (2008). "Tocilizumab for the treatment of rheumatoid arthritis." Expert Rev Clin Immunol 4(2): 165-172. Min, S. Y., M. Yan, Y. Du, T. Wu, E. Khobahy, S. R. Kwon, V. Taneja, A. Bashmakov, S. Nukala, Y. Ye, J. Orme, D. Sajitharan, H. Y. Kim and C. Mohan (2013). "Intra-articular nuclear factor-kappaB blockade ameliorates collagen-induced arthritis in mice by eliciting regulatory T cells and macrophages." Clin Exp Immunol 172(2): 217-227. Min, S. Y., M. Yan, S. B. Kim, S. Ravikumar, S. R. Kwon, K. Vanarsa, H. Y. Kim, L. S. Davis and C. Mohan (2015). "Green Tea Epigallocatechin-3-Gallate Suppresses Autoimmune Arthritis Through Indoleamine-2,3-Dioxygenase Expressing Dendritic Cells and the Nuclear Factor, Erythroid 2-Like 2 Antioxidant Pathway." J Inflamm (Lond) 12: 53. Minamino, T., H. Christou, C. M. Hsieh, Y. Liu, V. Dhawan, N. G. Abraham, M. A. Perrella, S. A. Mitsialis and S. Kourembanas (2001). "Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia." Proc Natl Acad Sci U S A 98(15): 8798-8803. Bibliografía 132 Miossec, P. and J. K. Kolls (2012). "Targeting IL-17 and TH17 cells in chronic inflammation." Nat Rev Drug Discov 11(10): 763-776. Mo, J., P. Panichayupakaranant, N. Kaewnopparat, A. Nitiruangjaras and W. Reanmongkol (2013). "Topical anti-inflammatory and analgesic activities of standardized pomegranate rind extract in comparison with its marker compound ellagic acid in vivo." J Ethnopharmacol 148(3): 901-908. Mohammad-Shahi, M., F. Haidari, B. Rashidi, A. A. Saei, S. Mahboob and M. R. Rashidi (2011). "Comparison of the effects of genistein and daidzein with dexamethasone and soy protein on rheumatoid arthritis in rats." Bioimpacts 1(3): 161-170. Mohammadshahi, M., F. Haidari, A. A. Saei, B. Rashidi, S. Mahboob and M. R. Rashidi (2013). "Soy protein, genistein, and daidzein improve serum paraoxonase activity and lipid profiles in rheumatoid arthritis in rats." J Med Food 16(2): 147-154. Molinero, L. L., A. Cubre, C. Mora-Solano, Y. Wang and M. L. Alegre (2012). "T cell receptor/CARMA1/NF-kappaB signaling controls T-helper (Th) 17 differentiation." Proc Natl Acad Sci U S A 109(45): 18529-18534. Montesinos, M. C., A. Desai, D. Delano, J. F. Chen, J. S. Fink, M. A. Jacobson and B. N. Cronstein (2003). "Adenosine A2A or A3 receptors are required for inhibition of inflammation by methotrexate and its analog MX-68." Arthritis Rheum 48(1): 240-247. Moon, S. J., J. S. Park, Y. J. Woo, M. A. Lim, S. M. Kim, S. Y. Lee, E. K. Kim, H. J. Lee, W. S. Lee, S. H. Park, J. H. Jeong, S. H. Park, H. Y. Kim, M. L. Cho and J. K. Min (2014). "Rebamipide suppresses collagen-induced arthritis through reciprocal regulation of th17/treg cell differentiation and heme oxygenase 1 induction." Arthritis Rheumatol 66(4): 874-885. Morel, J. and F. Berenbaum (2004). "Signal transduction pathways: new targets for treating rheumatoid arthritis." Joint Bone Spine 71(6): 503-510. Morinobu, A., W. Biao, S. Tanaka, M. Horiuchi, L. Jun, G. Tsuji, Y. Sakai, M. Kurosaka and S. Kumagai (2008). "(-)-Epigallocatechin-3-gallate suppresses osteoclast differentiation and ameliorates experimental arthritis in mice." Arthritis Rheum 58(7): 2012-2018. Motilva, V., E. Talero, J. R. Calvo, I. Villegas, C. Alarcon-de-la-Lastra and S. Sanchez-Fidalgo (2008). "Intestinal immunomodulation. Role of regulative peptides and promising pharmacological activities." Curr Pharm Des 14(1): 71-95. Mullazehi, M., L. Mathsson, J. Lampa and J. Ronnelid (2007). "High anti-collagen type-II antibody levels and induction of proinflammatory cytokines by anti-collagen antibody-containing immune complexes in vitro characterise a distinct rheumatoid arthritis phenotype associated with acute inflammation at the time of disease onset." Ann Rheum Dis 66(4): 537-541. Murakami, A., M. Song, S. Katsumata, M. Uehara, K. Suzuki and H. Ohigashi (2007). "Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation." Biofactors 30(3): 179-192. Musumeci, G., F. M. Trovato, K. Pichler, A. M. Weinberg, C. Loreto and P. Castrogiovanni (2013). "Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression." J Nutr Biochem 24(12): 2064-2075. Bibliografía 133 Nakayama, H., T. Yaguchi, S. Yoshiya and T. Nishizaki (2012). "Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner." Rheumatol Int 32(1): 151-157. Nandakumar, K. S., M. Andren, P. Martinsson, E. Bajtner, S. Hellstrom, R. Holmdahl and S. Kleinau (2003). "Induction of arthritis by single monoclonal IgG anti-collagen type II antibodies and enhancement of arthritis in mice lacking inhibitory FcgammaRIIB." Eur J Immunol 33(8): 2269- 2277. Nanes, M. S. (2003). "Tumor necrosis factor-alpha: molecular and cellular mechanisms in skeletal pathology." Gene 321: 1-15. Natarajan, V., B. Madhan and M. L. Tiku (2015). "Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics." PLoS One 10(6): e0127165. Nestorov, I. (2005). "Clinical pharmacokinetics of TNF antagonists: how do they differ?" Semin Arthritis Rheum 34(5 Suppl1): 12-18. Nie, H., Y. Zheng, R. Li, T. B. Guo, D. He, L. Fang, X. Liu, L. Xiao, X. Chen, B. Wan, Y. E. Chin and J. Z. Zhang (2013). "Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-alpha in rheumatoid arthritis." Nat Med 19(3): 322-328. Noack, M. and P. Miossec (2014). "Th17 and regulatory T cell balance in autoimmune and inflammatory diseases." Autoimmun Rev 13(6): 668-677. O'Dell, J. R., G. Paulsen, C. E. Haire, K. Blakely, W. Palmer, S. Wees, P. J. Eckhoff, L. W. Klassen, M. Churchill, D. Doud, A. Weaver and G. F. Moore (1999). "Treatment of early seropositive rheumatoid arthritis with minocycline: four-year followup of a double-blind, placebo-controlled trial." Arthritis Rheum 42(8): 1691-1695. Okada, Y., H. Nagase and E. D. Harris, Jr. (1987). "Matrix metalloproteinases 1, 2, and 3 from rheumatoid synovial cells are sufficient to destroy joints." J Rheumatol 14 Spec No: 41-42. Okamoto, H., T. Yoshio, H. Kaneko and H. Yamanaka (2010). "Inhibition of NF-kappaB signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritis." Arthritis Rheum 62(1): 82-92. Olsen, N. J. and C. M. Stein (2004). "New drugs for rheumatoid arthritis." N Engl J Med 350(21): 2167-2179. Omar, S. H. (2010). "Oleuropein in olive and its pharmacological effects." Sci Pharm 78(2): 133- 154. Pandey, K. B. and S. I. Rizvi (2009). "Plant polyphenols as dietary antioxidants in human health and disease." Oxid Med Cell Longev 2(5): 270-278. Park, C., D. O. Moon, I. W. Choi, B. T. Choi, T. J. Nam, C. H. Rhu, T. K. Kwon, W. H. Lee, G. Y. Kim and Y. H. Choi (2007). "Curcumin induces apoptosis and inhibits prostaglandin E(2) production in synovial fibroblasts of patients with rheumatoid arthritis." Int J Mol Med 20(3): 365-372. Park, J. S., S. K. Kwok, M. A. Lim, E. K. Kim, J. G. Ryu, S. M. Kim, H. J. Oh, J. H. Ju, S. H. Park, H. Y. Kim and M. L. Cho (2014). "STA-21, a promising STAT-3 inhibitor that reciprocally regulates Th17 and Treg cells, inhibits osteoclastogenesis in mice and humans and alleviates autoimmune inflammation in an experimental model of rheumatoid arthritis." Arthritis Rheumatol 66(4): 918- 929. Bibliografía 134 Park, J. S., M. A. Lim, M. L. Cho, J. G. Ryu, Y. M. Moon, J. Y. Jhun, J. K. Byun, E. K. Kim, S. Y. Hwang, J. H. Ju, S. K. Kwok and H. Y. Kim (2013). "p53 controls autoimmune arthritis via STAT-mediated regulation of the Th17 cell/Treg cell balance in mice." Arthritis Rheum 65(4): 949-959. Park, K. H., D. R. Gu, H. S. So, K. J. Kim and S. H. Lee (2015). "Dual Role of Cyanidin-3-glucoside on the Differentiation of Bone Cells." J Dent Res. Paul-Pletzer, K. (2006). "Tocilizumab: blockade of interleukin-6 signaling pathway as a therapeutic strategy for inflammatory disorders." Drugs Today (Barc) 42(9): 559-576. Peck, A. and E. D. Mellins (2009). "Breaking old paradigms: Th17 cells in autoimmune arthritis." Clin Immunol 132(3): 295-304. Pelucchi, C., C. Bosetti, M. Rossi, E. Negri and C. La Vecchia (2009). "Selected aspects of Mediterranean diet and cancer risk." Nutr Cancer 61(6): 756-766. Perez-Jimenez, F., G. Alvarez de Cienfuegos, L. Badimon, G. Barja, M. Battino, A. Blanco, A. Bonanome, R. Colomer, D. Corella-Piquer, I. Covas, J. Chamorro-Quiros, E. Escrich, J. J. Gaforio, P. P. Garcia Luna, L. Hidalgo, A. Kafatos, P. M. Kris-Etherton, D. Lairon, R. Lamuela-Raventos, J. Lopez-Miranda, F. Lopez-Segura, M. A. Martinez-Gonzalez, P. Mata, J. Mataix, J. Ordovas, J. Osada, R. Pacheco-Reyes, M. Perucho, M. Pineda-Priego, J. L. Quiles, M. C. Ramirez-Tortosa, V. Ruiz-Gutierrez, P. Sanchez-Rovira, V. Solfrizzi, F. Soriguer-Escofet, R. de la Torre-Fornell, A. Trichopoulos, J. M. Villalba-Montoro, J. R. Villar-Ortiz and F. Visioli (2005). "International conference on the healthy effect of virgin olive oil." Eur J Clin Invest 35(7): 421-424. Phillips, D. C., K. J. Woollard and H. R. Griffiths (2003). "The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species." Br J Pharmacol 138(3): 501-511. Pragasam, S. J., V. Venkatesan and M. Rasool (2013). "Immunomodulatory and antiinflammatory effect of p-coumaric acid, a common dietary polyphenol on experimental inflammation in rats." Inflammation 36(1): 169-176. Proudman, S. M., L. G. Cleland, R. G. Metcalf, T. R. Sullivan, L. D. Spargo and M. J. James (2015). "Plasma n-3 fatty acids and clinical outcomes in recent-onset rheumatoid arthritis." Br J Nutr 114(6): 885-890. Proudman, S. M., M. J. James, L. D. Spargo, R. G. Metcalf, T. R. Sullivan, M. Rischmueller, K. Flabouris, M. D. Wechalekar, A. T. Lee and L. G. Cleland (2015). "Fish oil in recent onset rheumatoid arthritis: a randomised, double-blind controlled trial within algorithm-based drug use." Ann Rheum Dis 74(1): 89-95. Puertollano, M. A., E. Puertollano, G. Alvarez de Cienfuegos and M. A. de Pablo Martinez (2010). "[Olive oil, immune system and infection]." Nutr Hosp 25(1): 1-8. Quemeneur, L., L. M. Gerland, M. Flacher, M. Ffrench, J. P. Revillard and L. Genestier (2003). "Differential control of cell cycle, proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides." J Immunol 170(10): 4986-4995. Ramadan, G. and O. El-Menshawy (2013). "Protective effects of ginger-turmeric rhizomes mixture on joint inflammation, atherogenesis, kidney dysfunction and other complications in a rat model of human rheumatoid arthritis." Int J Rheum Dis 16(2): 219-229. Ribbens, C., M. Martin y Porras, N. Franchimont, M. J. Kaiser, J. M. Jaspar, P. Damas, F. A. Houssiau and M. G. Malaise (2002). "Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment." Ann Rheum Dis 61(2): 161-166. Bibliografía 135 Ribel-Madsen, S., E. M. Bartels, A. Stockmarr, A. Borgwardt, C. Cornett, B. Danneskiold-Samsoe and H. Bliddal (2012). "A synoviocyte model for osteoarthritis and rheumatoid arthritis: response to Ibuprofen, betamethasone, and ginger extract-a cross-sectional in vitro study." Arthritis 2012: 505842. Rosillo, M. A., M. J. Alcaraz, M. Sanchez-Hidalgo, J. G. Fernandez-Bolanos, C. Alarcon-de-la-Lastra and M. L. Ferrandiz (2014). "Anti-inflammatory and joint protective effects of extra-virgin oliveoil polyphenol extract in experimental arthritis." J Nutr Biochem 25(12): 1275-1281. Rosillo, M. A., M. Sanchez-Hidalgo, A. Cardeno, M. Aparicio-Soto, S. Sanchez-Fidalgo, I. Villegas and C. A. de la Lastra (2012). "Dietary supplementation of an ellagic acid-enriched pomegranate extract attenuates chronic colonic inflammation in rats." Pharmacol Res 66(3): 235-242. Rosillo, M. A., M. Sanchez-Hidalgo, A. Cardeno and C. A. de la Lastra (2011). "Protective effect of ellagic acid, a natural polyphenolic compound, in a murine model of Crohn's disease." Biochem Pharmacol 82(7): 737-745. Rosillo, M. A., M. Sanchez-Hidalgo, A. Gonzalez-Benjumea, J. G. Fernandez-Bolanos, E. Lubberts and C. Alarcon-de-la-Lastra (2015). "Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis." Mol Nutr Food Res. Rosillo, M. A., M. Sanchez-Hidalgo, S. Sanchez-Fidalgo, M. Aparicio-Soto, I. Villegas and C. Alarcon-de-la-Lastra (2015). "Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen-induced arthritis." Eur J Nutr. Rubio, L., A. Macia, R. M. Valls, A. Pedret, M. P. Romero, R. Sola and M. J. Motilva (2012). "A new hydroxytyrosol metabolite identified in human plasma: hydroxytyrosol acetate sulphate." Food Chem 134(2): 1132-1136. Rudan, I., S. Sidhu, A. Papana, S. J. Meng, Y. Xin-Wei, W. Wang, R. M. Campbell-Page, A. R. Demaio, H. Nair, D. Sridhar, E. Theodoratou, B. Dowman, D. Adeloye, A. Majeed, J. Car, H. Campbell, W. Wang, K. Y. Chan and G. Global Health Epidemiology Reference (2015). "Prevalence of rheumatoid arthritis in low- and middle-income countries: A systematic review and analysis." J Glob Health 5(1): 010409. Sales, C., F. Oliviero and P. Spinella (2009). "[The mediterranean diet model in inflammatory rheumatic diseases]." Reumatismo 61(1): 10-14. Salgado, E. and J. R. Maneiro (2014). "[New therapies for rheumatoid arthritis]." Med Clin (Barc) 143(10): 461-466. Sanchez-Fidalgo, S., A. Cardeno, M. Sanchez-Hidalgo, M. Aparicio-Soto and C. A. de la Lastra (2013). "Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice." J Nutr Biochem 24(7): 1401-1413. Sanchez-Fidalgo, S., A. Cardeno, M. Sanchez-Hidalgo, M. Aparicio-Soto, I. Villegas, M. A. Rosillo and C. A. de la Lastra (2012). "Dietary unsaponifiable fraction from extra virgin olive oil supplementation attenuates acute ulcerative colitis in mice." Eur J Pharm Sci 48(3): 572-581. Sanchez-Fidalgo, S., A. Cardeno, M. Sanchez-Hidalgo, M. Aparicio-Soto, I. Villegas, M. A. Rosillo and C. A. de la Lastra (2013). "Dietary unsaponifiable fraction from extra virgin olive oil supplementation attenuates acute ulcerative colitis in mice." Eur J Pharm Sci 48(3): 572-581. Sanchez-Fidalgo, S., L. Sanchez de Ibarguen, A. Cardeno and C. Alarcon de la Lastra (2012). "Influence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model." Eur J Nutr 51(4): 497-506. Bibliografía 136 Sanchez-Fidalgo, S., I. Villegas, M. Aparicio-Soto, A. Cardeno, M. A. Rosillo, A. Gonzalez- Benjumea, A. Marset, O. Lopez, I. Maya, J. G. Fernandez-Bolanos and C. Alarcon de la Lastra (2015). "Effects of dietary virgin olive oil polyphenols: hydroxytyrosyl acetate and 3, 4- dihydroxyphenylglycol on DSS-induced acute colitis in mice." J Nutr Biochem 26(5): 513-520. Sanchez-Fidalgo, S., I. Villegas, A. Cardeno, E. Talero, M. Sanchez-Hidalgo, V. Motilva and C. Alarcon de la Lastra (2010). "Extra-virgin olive oil-enriched diet modulates DSS-colitis-associated colon carcinogenesis in mice." Clin Nutr 29(5): 663-673. Sanchez-Fidalgo, S., I. Villegas, M. A. Rosillo, M. Aparicio-Soto and C. A. de la Lastra (2015). "Dietary squalene supplementation improves DSS-induced acute colitis by downregulating p38 MAPK and NFkB signaling pathways." Mol Nutr Food Res 59(2): 284-292. Sanchez-Hidalgo, M., A. R. Martin, I. Villegas and C. Alarcon De La Lastra (2005). "Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces chronic colonic inflammation in rats." Biochem Pharmacol 69(12): 1733-1744. Sanchez-Hidalgo, M., A. R. Martin, I. Villegas and C. A. de la Lastra (2007). "Rosiglitazone, a PPARgamma ligand, modulates signal transduction pathways during the development of acute TNBS-induced colitis in rats." Eur J Pharmacol 562(3): 247-258. Sato, M., T. Miyazaki, F. Kambe, K. Maeda and H. Seo (1997). "Quercetin, a bioflavonoid, inhibits the induction of interleukin 8 and monocyte chemoattractant protein-1 expression by tumor necrosis factor-alpha in cultured human synovial cells." J Rheumatol 24(9): 1680-1684. Sawitzke AD, C. G. (2005). Immunomodulatory agents. Arthritis and allied conditions. Philadelphia, Lippincott, Williams and Wilkins: 885-914. Saxne, T. and D. Heinegard (1992). "Cartilage oligomeric matrix protein: a novel marker of cartilage turnover detectable in synovial fluid and blood." Br J Rheumatol 31(9): 583-591. Scotece, M., R. Gomez, J. Conde, V. Lopez, J. J. Gomez-Reino, F. Lago, A. B. Smith, 3rd and O. Gualillo (2012). "Further evidence for the anti-inflammatory activity of oleocanthal: inhibition of MIP-1alpha and IL-6 in J774 macrophages and in ATDC5 chondrocytes." Life Sci 91(23-24): 1229- 1235. Scott, D. L. and G. H. Kingsley (2006). "Tumor necrosis factor inhibitors for rheumatoid arthritis." N Engl J Med 355(7): 704-712. Schett, G. and S. L. Teitelbaum (2009). "Osteoclasts and Arthritis." J Bone Miner Res 24(7): 1142- 1146. Schett, G., M. Tohidast-Akrad, J. S. Smolen, B. J. Schmid, C. W. Steiner, P. Bitzan, P. Zenz, K. Redlich, Q. Xu and G. Steiner (2000). "Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis." Arthritis Rheum 43(11): 2501-2512. Schurgers, E., A. Billiau and P. Matthys (2011). "Collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-gamma." J Interferon Cytokine Res 31(12): 917- 926. Seong, A. R., J. Y. Yoo, K. Choi, M. H. Lee, Y. H. Lee, J. Lee, W. Jun, S. Kim and H. G. Yoon (2011). "Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-kappaB acetylation in fibroblast-like synoviocyte MH7A cells." Biochem Biophys Res Commun 410(3): 581-586. Bibliografía 137 Shakibaei, M., C. Buhrmann and A. Mobasheri (2011). "Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells." J Biol Chem 286(13): 11492- 11505. Shakibaei, M., G. Schulze-Tanzil, T. John and A. Mobasheri (2005). "Curcumin protects human chondrocytes from IL-l1beta-induced inhibition of collagen type II and beta1-integrin expression and activation of caspase-3: an immunomorphological study." Ann Anat 187(5-6): 487-497. Shin, G. C., C. Kim, J. M. Lee, W. S. Cho, S. G. Lee, M. Jeong, J. Cho and K. Lee (2009). "Apigenininduced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes." Chem Biol Interact 182(1): 29-36. Shishavan, N. G., B. P. Gargari, S. Kolahi, M. Hajialilo, M. A. Jafarabadi and Y. Javadzadeh (2015). "Effects of Vitamin K on Matrix Metalloproteinase-3 and Rheumatoid Factor in Women with Rheumatoid Arthritis: A Randomized, Double-Blind, Placebo-Controlled Trial." J Am Coll Nutr: 1- 7. Shouda, T., T. Yoshida, T. Hanada, T. Wakioka, M. Oishi, K. Miyoshi, S. Komiya, K. Kosai, Y. Hanakawa, K. Hashimoto, K. Nagata and A. Yoshimura (2001). "Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis." J Clin Invest 108(12): 1781-1788. Silva, S., B. Sepodes, J. Rocha, R. Direito, A. Fernandes, D. Brites, M. Freitas, E. Fernandes, M. R. Bronze and M. E. Figueira (2015). "Protective effects of hydroxytyrosol-supplemented refined olive oil in animal models of acute inflammation and rheumatoid arthritis." J Nutr Biochem 26(4): 360-368. Smedslund, G., M. G. Byfuglien, S. U. Olsen and K. B. Hagen (2010). "Effectiveness and safety of dietary interventions for rheumatoid arthritis: a systematic review of randomized controlled trials." J Am Diet Assoc 110(5): 727-735. Sun, Q. W., S. M. Jiang, K. Yang, J. M. Zheng, L. Zhang and W. D. Xu (2012). "Apigenin enhances the cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand in human rheumatoid arthritis fibroblast-like synoviocytes." Mol Biol Rep 39(5): 5529-5535. Sung, M. S., E. G. Lee, H. S. Jeon, H. J. Chae, S. J. Park, Y. C. Lee and W. H. Yoo (2012). "Quercetin inhibits IL-1beta-induced proliferation and production of MMPs, COX-2, and PGE2 by rheumatoid synovial fibroblast." Inflammation 35(4): 1585-1594. Suzuki, A. and K. Yamamoto (2015). "From genetics to functional insights into rheumatoid arthritis." Clin Exp Rheumatol 33(4 Suppl 92): 40-43. Suzuki, M., T. Tetsuka, S. Yoshida, N. Watanabe, M. Kobayashi, N. Matsui and T. Okamoto (2000). "The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF- alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts." FEBS Lett 465(1): 23-27. Symmons, D. P., M. Salmon, M. Farr and P. A. Bacon (1988). "Sulfasalazine treatment and lymphocyte function in patients with rheumatoid arthritis." J Rheumatol 15(4): 575-579. Thalhamer, T., M. A. McGrath and M. M. Harnett (2008). "MAPKs and their relevance to arthritis and inflammation." Rheumatology (Oxford) 47(4): 409-414. Tian, J., J. W. Chen, J. S. Gao, L. Li and X. Xie (2013). "Resveratrol inhibits TNF-alpha-induced IL- 1beta, MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway." Rheumatol Int 33(7): 1829-1835. Bibliografía 138 Tomita, T., Y. Kakiuchi and P. S. Tsao (2006). "THR0921, a novel peroxisome proliferatoractivated receptor gamma agonist, reduces the severity of collagen-induced arthritis." Arthritis Res Ther 8(1): R7. Tsao, R. (2010). "Chemistry and biochemistry of dietary polyphenols." Nutrients 2(12): 1231- 1246. Umar, S., A. Kumar, M. Sajad, J. Zargan, M. Ansari, S. Ahmad, C. K. Katiyar and H. A. Khan (2013). "Hesperidin inhibits collagen-induced arthritis possibly through suppression of free radical load and reduction in neutrophil activation and infiltration." Rheumatol Int 33(3): 657-663. van Everdingen, A. A., J. W. Jacobs, D. R. Siewertsz Van Reesema and J. W. Bijlsma (2002). "Lowdose prednisone therapy for patients with early active rheumatoid arthritis: clinical efficacy, disease-modifying properties, and side effects: a randomized, double-blind, placebo-controlled clinical trial." Ann Intern Med 136(1): 1-12. Vane, J. R. and R. M. Botting (1998). "Anti-inflammatory drugs and their mechanism of action." Inflamm Res 47 Suppl 2: S78-87. Vermeire, K., H. Heremans, M. Vandeputte, S. Huang, A. Billiau and P. Matthys (1997). "Accelerated collagen-induced arthritis in IFN-gamma receptor-deficient mice." J Immunol 158(11): 5507-5513. Verpoort, K. N., H. van Dongen, C. F. Allaart, R. E. Toes, F. C. Breedveld and T. W. Huizinga (2004). "Undifferentiated arthritis--disease course assessed in several inception cohorts." Clin Exp Rheumatol 22(5 Suppl 35): S12-17. Wang, J., Q. Zhang, S. Jin, D. He, S. Zhao and S. Liu (2008). "Genistein modulate immune responses in collagen-induced rheumatoid arthritis model." Maturitas 59(4): 405-412. Weber, S. M. and S. M. Levitz (2000). "Chloroquine interferes with lipopolysaccharide-induced TNF-alpha gene expression by a nonlysosomotropic mechanism." J Immunol 165(3): 1534-1540. Wegner, N., K. Lundberg, A. Kinloch, B. Fisher, V. Malmstrom, M. Feldmann and P. J. Venables (2010). "Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis." Immunol Rev 233(1): 34-54. Xiao, P., Y. Hao, X. Zhu and X. Wu (2013). "p53 contributes to quercetin-induced apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes." Inflammation 36(2): 272-278. Xie, C., L. Ma, J. Liu, X. Li, H. Pei, M. Xiang and L. Chen (2013). "SKLB023 blocks joint inflammation and cartilage destruction in arthritis models via suppression of nuclear factor-kappa B activation in macrophage." PLoS One 8(2): e56349. Xu, G., H. Nie, N. Li, W. Zheng, D. Zhang, G. Feng, L. Ni, R. Xu, J. Hong and J. Z. Zhang (2005). "Role of osteopontin in amplification and perpetuation of rheumatoid synovitis." J Clin Invest 115(4): 1060-1067. Xuzhu, G., M. Komai-Koma, B. P. Leung, H. S. Howe, C. McSharry, I. B. McInnes and D. Xu (2012). "Resveratrol modulates murine collagen-induced arthritis by inhibiting Th17 and B-cell function." Ann Rheum Dis 71(1): 129-135. Yamamoto, K., Y. Okada, A. Suzuki and Y. Kochi (2015). "Genetic studies of rheumatoid arthritis." Proc Jpn Acad Ser B Phys Biol Sci 91(8): 410-422. Yamanaka, H. (2015). "TNF as a target of inflammation in rheumatoid arthritis." Endocr Metab Immune Disord Drug Targets. Bibliografía 139 Yoon, C. H., S. J. Chung, S. W. Lee, Y. B. Park, S. K. Lee and M. C. Park (2013). "Gallic acid, a natural polyphenolic acid, induces apoptosis and inhibits proinflammatory gene expressions in rheumatoid arthritis fibroblast-like synoviocytes." Joint Bone Spine 80(3): 274-279. Yoon, H. Y., E. G. Lee, H. Lee, I. J. Cho, Y. J. Choi, M. S. Sung, H. G. Yoo and W. H. Yoo (2013). "Kaempferol inhibits IL-1beta-induced proliferation of rheumatoid arthritis synovial fibroblasts and the production of COX-2, PGE2 and MMPs." Int J Mol Med 32(4): 971-977. Youn, J., K. H. Lee, J. Won, S. J. Huh, H. S. Yun, W. G. Cho and D. J. Paik (2003). "Beneficial effects of rosmarinic acid on suppression of collagen induced arthritis." J Rheumatol 30(6): 1203-1207. Yun, H. J., W. H. Yoo, M. K. Han, Y. R. Lee, J. S. Kim and S. I. Lee (2008). "Epigallocatechin-3-gallate suppresses TNF-alpha -induced production of MMP-1 and -3 in rheumatoid arthritis synovial fibroblasts." Rheumatol Int 29(1): 23-29. Zhang, Y., J. Dong, P. He, W. Li, Q. Zhang, N. Li and T. Sun (2012). "Genistein inhibit cytokines or growth factor-induced proliferation and transformation phenotype in fibroblast-like synoviocytes of rheumatoid arthritis." Inflammation 35(1): 377-387. Zheng, Z., Y. Sun, Z. Liu, M. Zhang, C. Li and H. Cai (2015). "The effect of curcumin and its nanoformulation on adjuvant-induced arthritis in rats." Drug Des Devel Ther 9: 4931-4942. Zhu, L., J. Wang, T. Wei, J. Gao, H. He, X. Chang and T. Yan (2015). "Effects of Naringenin on inflammation in complete freund's adjuvant-induced arthritis by regulating Bax/Bcl-2 balance." Inflammation 38(1): 245-251. Zvaifler, N. J. (1965). "A Speculation on the Pathogenesis of Joint Inflammation in Rheumatoid Arthritis." Arthritis Rheum 8: 289-293.