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

Rosillo Ramírez, María de los Ángeles · 2016

Abstract

Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by inflammation of the synovial membrane and progressive destruction of the articular cartilage and bone. (Salgado and Maneiro 2014). RA patients exhibit an inflammatory chronic condition, which usually affects symmetrically arthrodial and small joints of hands and feet 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 et al. 2015). Although the specific triggers and exact mechanisms of tissue damage in RA is still unknown, an increase in inflammatory mediators as well as a dysregulation of the immune system with uncontrolled T cell activity, play a remarkable role in its pathogenesis. Pharmacological treatment in AR including nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) and biological agents have improved the signs and symptoms of RA, but these drugs are only effective in a fraction of patients and have other limitations including a high cost, the requirement for parenteral administration and important side effects. Therefore, new therapeutic strategies are under investigation including nutritional therapy. The beneficial effects of the Mediterranean diet have been proven not only in cardiovascular diseases but also in diabetes, obesity, arthritis and cancer (Cardeno, Sanchez-Hidalgo, and Alarcon-de-la-Lastra 2013). Evidence points out that Mediterranean diet decreases both pain and disease activity leading to better outcomes, and decreasing the doses of anti-inflammatory drugs, which exhibit important secondary effects (Smedslund et al. 2010). Olive Oil is the characteristic culinary fat of the Mediterranean area being described as a key bio-active food (Puertollano et al. 2010). Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree (Olea europea L.) solely by mechanical or other physical means under conditions that do not alter its natural composition. Traditionally the beneficial effects of EVOO have been ascribed to its monounsaturated fatty acid (MUFA) (Bermudez et al. 2011). However, a wide range of evidence indicates that 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. 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 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.

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

Author: Rosillo Ramírez, María de los Ángeles
Year: 2016
Source: https://idus.us.es/bitstreams/8b1c6342-cc27-4262-ab87-b827a5784156/download
DEPARTAMENTO DE FARMACOLOGÍA
FACULTAD DE FARMACIA
UNIVERSIDAD DE SEVILLA
FUNCIONALIDAD DEL ACEITE DE OLIVA
VIRGEN EXTRA EN LA ARTRITIS
REUMATOIDE EXPERIMENTAL
Tesis Doc o al p esen ada po
MARÍA DE LOS ÁNGELES ROSILLO RAMÍREZ
Pa a op a al G ado de Doc o a en Fa macia
Se illa, 2015
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DEPARTAMENTo D , FAnMACoLociA
FACULTAD DE FARMACIA
UNTVERSIDAD DE SEVILLA
Las D as. Ca alina Ala cón de la Las a Rome o, Ca ed á ica de Uni e sidad y
Ma ina Sánchez Hidalgo, P o eso a Con a ada Doc o a, adsc i as al Depa amen o de
Fa macología de la Facul ad de Fa macia de la Uni e sidad de Se illa.
IN FORMAN
QUE IA TCSiS DOCIOTAI i UIAdA "FUNCIONALIDAD DE ACEITE DE OLIVA
VIRGEN EXTRA EN LA ARRITIS REUMATOIDE EXPERIMENTAL" p esen ada po la Lda.
Ma ía de los Ángeles Rosillo Ramí ez pa a op a al g ado de Doc o a en Fa macia con
Mención ln e nacional ha sido lle ada a cabo bajo su di ección.
Se illa, 16 de no iemb e de 2015
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D a. Ca alina Ala cón de la Las a Rome o D a. Ma ina Sánchez Hidalgo
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DEPARTAMENTO DE FARMACOLOGiA
FACULTAD DE FARMACIA
I]N T!'ERSID{) DE SDI'ILLA
Dña. ME Dolo es Ga cía Giménez, P o eso a Ca ed á ¡ca de Fa macología y Di ec o a
del Depa amen o de Fa macología de Ia Facul ad de Fa macia de la Uni e sidad de
Se illa.
CERTIFICA:
Que la Tesis Doc o al i ulada "FUNCIONALIDAD DEL ACEITE DE OIIVA VIRGEN EXTRA
EN A ARRITIS REUMATOIDE EXPERIMENTAL" ealizada po la Lda. Ma ía de los
Ángeles Rosillo Ramí ez, ha sido di igida po la D a. Ca al¡na Ala cón de la Las a
Rome o y la D a. Ma ¡na Sánchez Hidalgo, pa a aspi a al g ado de Doc o en Fa macia
con Mención ln e nac¡onal, cumpliendo los equisi os pa a es e ipo de abajo.
Y pa a que asícons e, i mo la p esen e.
En Se illa, a 16 de no iemb e de 2013
D a. Me Dolo es Ga cía
Es e abajo de in es igación se ha lle ado a cabo g acias a la inanciación de las
siguien es ins i uciones y p oyec o de in es igación:
PROYECTO DE INVESTIGACIÓN
‒ Valo ación del Acei e de Oli a Vi gen Ex a en la A i is Reuma oide Expe imen al:
Es udio Biodi igido, Ca ac e ización Fa macológica y Desa ollo de Ing edien es
Funcionales.
En idad inanciado a: Conseje ía de Inno ación, Ciencia y Emp esa. Jun a de Andalucía.
In es igado p incipal: Ca alina Ala cón de la Las a Rome o.
Re e encia: AGR-6609
Du ación: 2011- 2016
BECA PREDOCTORAL
Beca p edoc o al asociada a P oyec os de In es igación de Excelencia en Equipos de
In es igación O den de 11 de Diciemb e de 2007 (BOJA 4, 05/01/2008) Con oca o ia
2010 (BOJA 135, 12/07/2011).
En idad inanciado a: Conseje ía de Inno ación, Ciencia y Emp esa. Jun a de Andalucía.
Y g acias a las es ancias ealizadas en:
‒ Luga : Depa amen o de Fa macología. Facul ad de Fa macia. Uni e sidad de Valencia,
España.
Du ación: Oc ub e-No iemb e 2012.
In es igado esponsable: Ma ía José Alca az To mo.
‒ Luga : Depa amen o de Reuma ología. E asmus MC. Uni e sidad E asmus. Ro e dam,
Holanda
Du ación: Sep iemb e-Diciemb e 2014.
In es igado esponsable: E ik Lubbe s.
Publicaciones
Ma ía de los Ángeles Rosillo Ramí ez, Licenciada en Fa macia po la Uni e sidad de
Se illa en 2009. En 2010 cu só el Más e en A ención Fa macéu ica y Fa macia Asis encial en la
misma uni e sidad, du an e es e año ecibió una beca de Plan de Apoyo al Más e O icial de la
Uni e sidad de Se illa. Ese mismo año consiguió el P emio a la mejo Beca sob e “Tema Lib e”
“Ac i idad del zumo de la g anada y sus componen es en la in lamación c ónica in es inal
asociada a cánce colo ec al expe imen al” concedido po la Fundación Fa macéu ica A enzoa .
Desde el año 2010 o ma pa e del g upo de in es igación Fa macología Expe imen al y
Fa macia Clínica (CTS-259). Comenzó sus es udios de Doc o ado en la Uni e sidad de Se illa en
el año 2011. En 2012 ob u o una beca p edoc o al Adsc i a a P oyec os de In es igación de
Excelencia en Equipos de In es igación asociada al p oyec o AGR 6609 de la Conseje ía de
Economía, Inno ación y Ciencia de la Jun a de Andalucía pa a la ealización de su Tesis Doc o al
bajo la di ección de las doc o as Ca alina Ala cón de la Las a Rome o y Ma ina Sánchez Hidalgo.
Comple ó su o mación p edoc o al en el Depa amen o de Fa macología de la
Uni e sidad de Valencia bajo la di ección de la doc o a Mª José Alca az To mo du an e los meses
de oc ub e y no iemb e de 2012 y en el Depa amen o de Reuma ología e Inmunología del
E asmus Medical Cen e de Ro e dam (Holanda) bajo la di ección del doc o E ik Lubbe s
du an e los meses de sep iemb e a diciemb e de 2014.
Ha pa icipado en dis in os p oyec os, así como en di e sos abajos de in es igación
que han sido p esen ados en cong esos nacionales e in e nacionales y que han dado luga a
dis in as publicaciones en e is as in e nacionales.
PROYECTOS DE INVESTIGACIÓN
‒ Valo ación del Acei e de Oli a Vi gen Ex a en la A i is Reuma oide Expe imen al:
Es udio Biodi igido, Ca ac e ización Fa macológica y Desa ollo de Ing edien es
Funcionales (AGR-6609 – Beca ia p edoc o al) Jun a de Andalucía. In es igado
p incipal: D a. Ca alina Ala cón de la Las a Rome o.

Publicaciones
PUBLICACIONES EN REVISTAS INTERNACIONALES
De i adas de la esis:
‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fe nández-Bolaños JG, Lubbe s
E, Ala cón-de-la-Las a C. P e en i e e ec s o die a y hyd oxy y osol ace a e, an ex a
i gin oli e oil polyphenol in mu ine collagen-induced a h i is. Mol Nu Food 2015. In
p ess.
‒ Rosillo MA, Sánchez-Hidalgo M, Sánchez-Fidalgo S, Apa icio-So o M, Villegas I, Ala cón-
de-la-Las a C. Die a y ex a- i gin oli e oil p e en s in lamma o y esponse and
ca ilage ma ix deg ada ion in mu ine collagen-induced a h i is. Eu J Nu . 2015. In
p ess.
‒ Rosillo MA, Alca az MJ, Sánchez-Hidalgo M, Fe nández-Bolaños JG, Ala cón-de-la-Las a
C, Fe ándiz ML. An i-in lamma o y and join p o ec i e e ec s o ex a- i gin oli e-oil
polyphenol ex ac in expe imen al a h i is. J Nu Biochem. 2014;25(12):1275-81.
No elacionadas con la esis:
‒ Apa icio-So o M, Sánchez-Hidalgo M, Cá deno A, Rosillo MA, Sánchez-Fidalgo S, U illa
J, Ma ín-Laca e I, Ala cón-de-la-Las a C. Die a y ex a i gin oli e oil a enua es kidney
inju y in p is ane-induced SLE model ia ac i a ion o HO-1/N -2 an ioxidan pa hway
and supp ession o JAK/STAT, NF-κB and MAPK ac i a ion. J Nu Biochem. 2015. In
p ess.
‒ Sánchez-Fidalgo S, Villegas I, Apa icio-So o M, Cá deno A, Rosillo MA, González-
Benjumea A, Ma se A, López Ó, Maya I, Fe nández-Bolaños JG, Ala cón de la Las a C.
E ec s o die a y i gin oli e oil polyphenols: hyd oxy y osyl ace a e and 3, 4-
dihyd oxyphenylglycol on DSS-induced acu e coli is in mice. J Nu Biochem.
2015;26(5):513-20.
‒ Sánchez-Fidalgo S, Villegas I, Rosillo MA, Apa icio-So o M, de la Las a CA. Die a y
squalene supplemen a ion imp o es DSS-induced acu e coli is by down egula ing p38
MAPK and NFkB signaling pa hways. Mol Nu Food Res. 2015;59(2):284-92.
Publicaciones
‒ González-Mau aza H, Ma ín-Co de o C, Ala cón-de-la-Las a C, Rosillo MA, León-
González AJ, Sánchez-Hidalgo M. An i-in lamma o y e ec s o Re ama monospe ma in
acu e ulce a i e coli is in a s. J Physiol Biochem. 2014;70(1):163-72.
‒ Cá deno A, Sánchez-Hidalgo M, Rosillo MA, Ala cón de la Las a C. Oleu opein, a
secoi idoid de i ed om oli e ee, inhibi s he p oli e a ion o human colo ec al cance
cell h ough down egula ion o HIF-1α. Nu Cance . 2013;65(1):147-56.
‒ Sánchez-Fidalgo S, Cá deno A, Sánchez-Hidalgo M, Apa icio-So o M, Villegas I, Rosillo
MA, de la Las a CA. Die a y unsaponi iable ac ion om ex a i gin oli e oil
supplemen a ion a enua es acu e ulce a i e coli is in mice. Eu J Pha m Sci.
2013;48(3):572-81.
‒ Rosillo MA, Sánchez-Hidalgo M, Cá deno A, Apa icio-So o M, Sánchez-Fidalgo S, Villegas
I, de la Las a CA. Die a y supplemen a ion o an ellagic acid-en iched pomeg ana e
ex ac a enua es ch onic colonic in lamma ion in a s. Pha macol Res. 2012;66(3):235-
42.
‒ Rosillo MA, Sánchez-Hidalgo M, Cá deno A, de la Las a CA. P o ec i e e ec o ellagic
acid, a na u al polyphenolic compound, in a mu ine model o C ohn's disease. Biochem
Pha macol. 2011;82(7):737-45.
COMUNICACIONES A CONGRESOS
De i adas de la esis:
In e nacionales
‒ Rosillo MA, Alca áz MJ, Fe ándiz, ML, Sánchez-Hidalgo M, Sánchez-Fidalgo S, Villegas I,
Ala cón-de-la-Las a C. “POLYPHENOL EXTRACT FROM EXTRA VIRGIN OLIVE OIL
AMELIORATES TYPE II COLLAGEN-INDUCED ARTHRITIS IN DBA/1 J MICE” Pós e . 13 h
ISANH In e na ional Con e ence on An ioxidan s. Ma akech (Mo occo), Junio de 2013.
Nacionales
Publicaciones
‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fe nández-Bolaños JG, Méndez-
Gu ié ez A, Pé ez-Domíngez M, Villegas I, Lubbe s E, Ala cón-de-la-Las a C. DIETARY
HYDROXYTYROSOL ACETATE PREVENTS INFLAMMATORY RESPONSE AND JOINT
DAMAGE IN MURINE EXPERIMENTAL ARTHRITIS. Comunicación o al p emiada. 36
Cong eso de la Sociedad Española de Fa macología. Valencia, Sep iemb e de 2015.
‒ Rosillo MA, Sánchez-Hidalgo M, González-Benjumea A, Fe nández-Bolaños JG, Méndez-
Gu ié ez A, Pé ez-Domíngez M, Villegas I, Lubbe s E, Ala cón-de-la-Las a C. ANTI-
INFLAMMATORY EFFECTS AND JOINT PROTECTION OF A HYDROXYTYROSOL ACETATE
ENRICHED DIET, AN EXTRA VIRGIN OLIVE OIL POLYPHENOL, IN COLLAGEN-INDUCED
ARTHRITIS IN MICE. Comunicación o al. VII Jo nadas de Jó enes Fa macólogos de
Andalucía. G anada, Julio de 2015.
‒ Rosillo MA, Fe ándiz ML, Sánchez-HidalgoML, Fe nández-Bolaños JG, Ala cón-de-la-
Las a C, Alca az MJ. ANTI-INFLAMMATORY AND JOINT PROTECTIVE EFFECTS OF A
POLYPHENOLIC EXTRACT FROM EXTRA VIRGIN OLIVE OIL IN TYPE II COLLAGEN-INDUCED
ARTHRITIS. Pós e . XXXV Cong eso de la Sociedad Española de Fa macología. Mad id,
Sep iemb e de 2014.
‒ Rosillo MA, Méndez-Gu ié ez A, Fe andiz ML, Sánchez-Hidalgo M, Fe nández-Bolaños
JG, Ala cón-de-la-Las a C, Alca az MJ. ANTI-IFLAMMATORY AND JOINT PROTECTIVE
EFFECTS OF EXTRA VIRGIN OLIVE OIL POLYPHENOL EXTRACT IN EXPERIMENTAL
ARTHRITIS. Comunicación o al. VI Jo nadas de Jó enes Fa macólogos de Andalucía.
Se illa, Junio de 2014.
No elacionados con la esis:
In e nacionales
‒ Rosillo MA, Sánchez-Hidalgo M, Cá deno A, Apa icio-So o M, Conde M, Sánchez-Fidalgo
S, Villegas I, Ala cón-de-la-Las a C. BENEFICIAL EFFECTS OF AND ELLAGIC ACID-
ENRICHED POMEGRANATE EXTRACT ON CHRONIC TNBS-INDUCED COLITIS IN RATS.
Pós e . 6 h Eu opean Cong ess o Pha macology. G anada (Spain), Julio de 2012.
‒ Sánchez-Fidalgo S, Cá deno A, Apa icio-So o M, Villegas I, Sánchez-Hidalgo M, Rosillo
MA, Ala cón-de-la-Las a C. EFFECTS OF DIETARY UNSAPONIFIABLE FRACTION FROM
Publicaciones
EXTRA VIRGIN OLIVE OIL ON ACUTE ULCERATIVE COLITIS. Pós e . 20 h Uni ed Eu opean
Gas oen e ology Week (UEGW) Ams e dam, Oc ub e de 2012. GUT: BMJ; 2012.
‒ Sánchez-Fidalgo S, Cá deno A, Apa icio-So o M, Sánchez-Hidalgo M, Rosillo MA, Villegas
I, Ala cón-de-la-Las a C. DIETARY EXTRA VIRGIN OLIVE OIL POLPHENOLS
SUPLEMENTATION MODULATES DSS-INDUCED CHRONIC COLITIS IN MICE. Pós e . 20 h
Uni ed Eu opean Gas oen e ology Week (UEGW) Ams e dam, Oc ub e de 2012. GUT:
BMJ; 2012.
‒ Pa as-Cha ez C, Masoodi M, Sánchez-Hidalgo M, Noakes P, Ala cón-de-la-Las a C,
Rosillo MA. THE ROLE OF NULEAR FACTOR κβ ON THE SYNTHESIS OF LIPID
INFLAMMATORY MEDIATORS ON THP-1 DERIVED MACROPHAGES. Pós e . 10 h
Cong ess o he In e na ional Socie y o he S udy o Fa y Acids and Lipids. Vancou e ,
2012.
‒ Cá deno A, Sánchez-Hidalgo M, Rami ez E, Apa icio-So o M, Rosillo MA, Ala cón-de-la-
Las a C. NATURAL DIETARY POLYPHENOL, ELLAGIC ACID SUPRESSES COX-2 EXPRESSION
IN HUMAN MONOCYTE THP-1 MACROPHAGES THROUGH MAPKs PATHWAYS. Pós e .
19 h Uni ed Eu opean Gas oen e ology Week (UEGW). S ockholm, Oc ub e de 2011.
GUT 2011; 60 (Suppl 3) A1.
‒ González-Mau aza NH, Sánchez-Hidalgo M, Ma ín-Co de o C, Rosillo MA, Tale o E,
Ala cón-de-la-Las a C. RETAMA MONOSPERMA (L.) BOISSIER AQUEOUS EXTRACT
ATTENUATES COLONIC INFLAMATION IN AN EXPERIMENTALLY ACUTE TNBS-INDUCED
COLITIS MODEL. Comunicación o al. 18 h Uni ed Eu opean Gas oen e ology Week
(UEGW) Ba celola, Oc ub e de 2010. GUT:BMJ; 2010.
‒ Rosillo MA, Sánchez-Hidalgo M, Cá deno A, Ala cón-de-la-Las a C. THE EFFECTS OF
ELLAGIC ACID, A POLYPHENOL DERIVED FROM POMEGRANATE, IN A EXPERIMENTALLY
ACUTE TNBS-INDUCED COLITIS MODEL. Pós e . 18 h Uni ed Eu opean Gas oen e ology
Week (UEGW) Ba celola, Oc ub e de 2010. GUT:BMJ; 2010.
Nacionales
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3
1. Rheuma oid A h i is
Rheuma oid a h i is (RA) can be de ined as a ch onic in lamma o y disease wi h
sys emic au oimmune componen , and is mainly cha ac e ized by agg essi e syno ial
hype plasia, syno i is, p og essi e des uc ion o ca ilage, and bone e osion wi h pain ul
swelling o small join s, a igue, p olonged s i ness, and e e caused by immune esponses and
speci ic inna e in lamma o y p ocesses (Z ai le 1965, C eeme s 2004, B ooks 2006).
Global p e alence o RA has been es ima ed o be a ound 0.5-1.0% o adul s in
de eloped coun ies wi h a la ge a ia ion ac oss egions and app oxima ely h ee- imes mo e
common in he emale gende . The disease may begin a any age, bu a ound 80% o all pa ien s
ini ia e he disease be ween he ages o 35 and 50 yea s (Rudan, Sidhu e al. 2015).
RA pa ien s exhibi an in lamma o y ch onic condi ion, which usually a ec s
symme ically dia h odial and small join s o hands and ee . RA is cha ac e ized by he
in lamma ion o syno ial join issues leading hype plasia o he syno ial lining cell laye and he
o ma ion o heuma oid pannus, which is capable o des oying adjacen ca ilage and bone
and causing subsequen join de o mi y (Ve poo , an Dongen e al. 2004). Addi ional
cha ac e is ic ea u es o RA syno i is include neo-angiogenesis and in il a ion o immune cells
such as mac ophages and lymphocy es which can o m agg ega es. Au oimmuni y, iden i ied by
he p oduc ion o au o-an ibodies such as heuma oid ac o (RF) o an i-ci ullina ed p o ein
an ibodies (ACPA) p ecedes he clinically de ec able onse o in lamma o y a h i is and can las
o yea s (Chapuy-Regaud, Noguei a e al. 2005, Wegne , Lundbe g e al. 2010). The clinic
ou come is ex emely a iable and may ex end om a so au olimi an AR o a mul isys emic
in lamma ion wi h a apid ou come (Finckh 2009). In his sense, abou 40% o pa ien s, AR
disease may in ol e an ex aa icula componen , a ec ing a ious o gans and sys ems
including skin, lungs, eyes, hea and blood essels. The p og essi e a icula o ex a-a icula
de e io a ion, leads de o mi y, pain, unc ional disabili y and a dec ease in li e expec ancy o

In oducción
4
hese pa ien s. These cha ac e is ics jus i y i s mul idisciplina y app oach, which includes
medical ea men , physio he apy and nu i ional he apy. The di e en ea men s a e aimed
p ima ily o supp ess he in lamma o y p ocess alle ia ing he symp oms and signs o he
disease.
The e iology o RA is unknown. A gene ic p edisposi ion sugges ed by a link wi h HLA-
DR4 and ela ed allo ypes o MHC class II, he T-cell-associa ed p o ein PTPN22, STAT4 and
TRAFI1/C5, among o he s gene polymo phisms, has been ela ed o a wo se ou coming and a
highe deg ee o a icula des uc ion and ex a-a icula mani es a ions (Messemake , Huizinga
e al. 2015). Howe e , he conco dance a e o RA in monozygo ic wins is low bu highe han
ha in dizygo ic wins sugges ing ha in addi ion o gene ic ac o s, en i onmen al ac o s as
well as hei in e ac ion can be decisi e in he de elopmen and disease p og ession. Among
en i onmen al ac o s, smoking has been ecognized as a majo isk ac o RA. In ac , smoking
has been associa ed wi h an inc eased isk o de eloping se oposi i e RA (RF and/o ACPA)
(Chang, Yang e al. 2014). Ne e heless, o he en i onmen al ac o s including socio-economic,
ho monal, silica exposu e, s imuli like i uses, bac e ia and s ess, and die e ic ac o s may
con ibu e o he disease p og ession (Cu olo 2007, Gomez-Pue a, Gedmin as e al. 2013).
The pa hogenesis o RA is incomple ely unde s ood bu hype plasia o he syno ial
memb ane is a hallma k o RA pa hology, which is cha ac e ized by bo h hype p oli e a ion o
syno ial ib oblas s and massi e in il a ion o in lamma o y immune cells, including CD4+ T-
cells and inna e immune cells (Koma su and Takayanagi 2012). Once he p ocess s a s, a
p og essi e ec ui men o in lamma o y T cells and mac ophages in o he join s occu s h ough
a complex se ies o adhesion and mig a o y e en s. In his sense, ini ially he e is an ex ensi e
inc ease in he numbe o inna e e ec o cells in he syno ial lining laye , which becomes se e al
laye s hick. Sublining laye becomes in il a ed wi h in lamma o y mononuclea cells, including
lymphocy es and mac ophages and mas cells. In ac , mac ophages a e cen al e ec o s o
syno i is and ac hough elease o cy okines, eac i e oxygen and ni ogen in e media es,
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5
p oduc ion o p os anoids and ma ix-deg ading enzymes, phagocy osis and an igen
p esen a ion. In addi ion, neu ophils ansmig a e apidly om he in a ascula compa men
o syno ial luid in he join ca i y con ibu ing o he join swelling and syn hesizing
p os aglandins, p o eases and eac i e oxygen in e media es (Cascao, Rosa io e al. 2010). All o
hese cells, including esiden ib oblas s, p oduce cy okines, which oge he wi h locally
p oduced au oan ibodies and immune complexes and complemen , main ain he ch onic
in lamma ion leading he memb ane expansion, which o ms pannus. The cell laye o ib oblas -
and mac ophage-like cells which compose he pannus, can p oduce p o einases, ecep o
ac i a o o nuclea ac o kappa B ligand (RANKL) and o he ac o s causing ca ilage
des uc ion. The pe sis en syn hesis o p oin lamma o y cy okines plays a ema kable ole in
he de elopmen o RA disease om ea ly au oimmuni y s ep h ough con inuous ch onic
in lamma ion in o des uc ion o join issue. In addi ion, he p olonged immune ac i a ion is
he main esponsible o e osi e and sys emic bone loss and an inc eased p opensi y o all in RA
pa ien s.
The de elopmen o he in lamma o y p ocess in RA in ol es many di e en ype o cells
and a complex cy okine ne wo k. Upon ac i a ion and expansion, CD4
+
T cells de elop in o
di e en T helpe cell subse s wi h di e en cy okine p o ile and dis inc e ec o unc ions.
Ac i a ed T cells ha sec e IFNϒ, IL2, IL12, IL18, TNF a e p oduced in he syno ial luid and
exp essed in he syno ial memb ane. Pa icula ly, TNF and IL-1β a e conside ed as he main
p oin lamma o y cy okines in he pa hogenesis o RA. TNF plays a undamen al ole h ough
ac i a ion o cy okine and chemokine exp ession, exp ession o endo helial-cell adhesion
molecules, p o ec ion o syno ial ib oblas s, p omo ion o angiogenesis, supp ession o
egula o y T cells and induc ion o pain (McInnes and Sche 2011). In ac , ea men wi h TNF-
α inhibi o s esul s in dec eased in lamma ion and bone p o ec ion in RA pa ien s (Sco and
Kingsley 2006). IL-1 amily cy okines a e also abundan ly exp essed in RA and p omo e he
ac i a ion o leucocy es, endo helial cells, chond ocy es and os eoclas s (B ennan and McInnes
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6
2008). Th17 cells, ia hei p oduc ion o IL-17, con ibu e c ucially o join in lamma ion and
bone e osion in RA (Ko ake, Udagawa e al. 1999, Lubbe s 2010, Jung, Kim e al. 2014). IL-6 and
TGF-β induce Th17 de elopmen and IL-23 p omo es Th17 cell expansion. In addi ion, hese
cy okines ac i a e mac ophages o sec e o he p oin lamma o y cy okines such as TNF and IL-
1β, IL-6, IL-12, RANKL exp ession on T cells, p omo e he di e en ia ion o B cells and s imula e
he elease o ma ix me allop o eases (MMP) p o oking he deg ada ion o he ca ilage and
he ac i a ion o os eoclas s leading o he bone eso p ion (Sche and Tei elbaum 2009,
Chimen i, T iggianese e al. 2015).
Figu e 1. Adap i e and Inna e Immune P ocesses wi hin he Join in Rheuma oid A h i is (McInnes and
Sche 2011)
Unde s anding he in acellula a ge s ha egula e cy okines in RA can po en ially lead
o new he apeu ic in e en ions. In his sense, he inhibi ion o key signal ansduc ion
pa hways in in lamma ion such as mi ogen-ac i a ed p o ein kinase (MAPK) pa hway,
phospha idylinosi ol-3 p o ein kinase (PI3) pa hway, janus kinase-signal ansduce and
ac i a o o ansc ip ion (Jak-STAT) and nuclea ac o kappa B (NFKB) would be expec ed o
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7
abolish he cell ac i a ion by cy okines and p oduc ion o hese p oin lamma o y cy okines in
RA (Mo el and Be enbaum 2004). Fo example, NF-κB is ac i a ed in he syno ium o pa ien s
wi h RA and egula es genes ha con ibu e o in lamma ion including TNF, IL-6, IL8, iNOS and
COX-2 (Auppe le, Benne e al. 2001). Simila ly, MAPK ha e a ac ed conside able a en ion as
po en he apeu ic a ge s in RA. MAPK a e also key egula o s o cy okines and MMP
p oduc ion and could also a ge ed in RA. All h ee kinases amilies, c-Jun N- e minal kinase
(JNK), p38 and ex acellula signal- egula ed kinase (ERK), a e exp essed in heuma oid syno ial
issue (Sche , Tohidas -Ak ad e al. 2000).
1.1. Pha macological ea men o RA
The cu en pha macological he apies o he ea men o RA a e designed o as
con ol he disease ac i i y and p e en he p og ession o damage in issues. These include
ea ly and s ic con ol o he ac i i y, he use o d ugs o he ea men o mode a e disease,
and induc ion o emission wi h use o biological d ugs.
Fi s line he apy including medica ions ha supp ess in lamma ion such as nons e oidal
an i-in lamma o y d ugs (NSAIDs) and glucoco icoids; hey ac apidly o imp o e pain and
swelling due o RA. Disease-modi ying an i heuma ic d ugs (DMARDs) a e slowe -ac ing
compounds, no only imp o e symp oms bu also diminish clinical and adiog aphic p og ession.
In 1999, i s biological- esponse modi ie was ma ke ed. These agen s we e designed o a ge
he in lamma o y media o s o issue damage in RA (TNF-α, IL-1). The newes pha macological
a ge a e an i-IL-6- ecep o monoclonal an ibodies.
NSAIDs
NSAIDs elie e he bone and join in lamma o y symp oms o RA; can be use ul in he
i s weeks a e he onse o RA symp oms while a diagnos ic wo kup is unde aken, be o e a
diagnosis is ce ain, and as b idge he apy while wai ing o a slow-ac ing DMARD o become
e ec i e. The mechanism o ac ion o NSAIDs is he inhibi ion o COX. COX is c i ical in he
me abolism o a achidonic acid o he p o-in lamma o y p os aglandins. Two iso o ms o COX
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8
ha e been ex ensi ely s udied in humans: COX-1 and COX-2. COX-1 is exp essed cons i u i ely
in he s omach, in es ines, kidneys, and pla ele s and is in ol ed in unc ions such as gas ic
p o ec ion om hyd ochlo ic acid. COX-2, a p edominan ly inducible isoenzyme, is in ol ed in
he p oduc ion o p os aglandins E2 and I2, which a e up egula ed in he in lamma o y esponse
(Vane and Bo ing 1998). NSAIDs a e usually well ole a ed o sho pe iods o ime; howe e ,
wi h ch onic use hey some imes lead o gas oin es inal complica ions, such us ulce o ma ion,
pe o a ions, and bleeding (Chiba, Sa o e al. 2005). Ano he po en ial ad e se e ec o NSAIDs
is hei enal oxici y.
Glucoco icoids.
Glucoco icoids ha e been widely used in he ea men o RA. Al hough high doses o
glucoco icoids clea ly cause unaccep able oxici y, daily doses o p ednisone (o i s equi alen )
o ≤15 mg may diminish pain and swelling in many pa ien s. This dosage o glucoco icoids may
also ha e a limi ed disease-modi ying e ec in pa ien s wi h RA ( an E e dingen, Jacobs e al.
2002). Fi s , hey p omo e he exp ession o lipoco in-1, which inhibi s he enzyme
phospholipase A2 and he gene a ion o a achidonic acid. Second, hei ansc ip ion p oduc s
inhibi he ac ion o NF-κΒ and AP-1 p o ein, which ac o up egula e p oin lamma o y
molecules, like TNF and IL-1. Thi d, hey exe an e ec h ough memb ane-associa ed ecep o s
and second messenge s-a nongenomic e ec (De Bossche , Schmi z e al. 1997). Glucoco icoids
p esen a la ge numbe o ad e se e ec s such us, diabe es, hype ension, pep ic ulce , among
o he s. Gi en he slow onse o ac ion o adi ional DMARDs, low doses o glucoco icoids a e
o en used as a b idge he apy o con ol symp oms un il he DMARDs o biological agen s
become e ec i e. Mono he apy wi h glucoco icoids is no ecommended.
DMARDs
Me ho exa e
Me ho exa e low dose is used as an immunosupp essan in au oimmune and heuma ic
diseases. Me ho exa e ea men has demons a ed e ec i eness, eliabili y, sus ained long-

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9
e m ac ion and high ole abili y. Up o ou mechanisms o ac ion ha e been p oposed o he
d ug. Fi s , i wo ks as an an i ola e agen (Quemeneu , Ge land e al. 2003). Second,
me ho exa e may diminish he accumula ion o oxic compounds, especially polyamines, which
con ibu e o issue inju y in RA (C ons ein 2005). Thi d, me ho exa e may educe in acellula
le els o glu a hione (Phillips, 2003). Finally, i inc eases ex acellula adenosine le els, an an i-
in lamma o y ac ion (Mon esinos, Desai e al. 2003). All o hese mechanisms induce inhibi ion
o he immune cellula p oli e a ion and s ong an i-in lamma o y ac i i y. Po en ial ad e se
e ec s o me ho exa e include hepa i is and ci hosis, o al ulce s, cy openias, and in e s i ial
pneumoni is. Acco ding hese side e ec s, i is impo an moni o ing pa ien s ecei ing
me ho exa e ea men (K eme , Ala con e al. 1994).
Hyd oxychlo oquine
Hyd oxychlo oquine is an an imala ial ha has been in oduced as he apy o RA. The
e icacy o hyd oxychlo oquine depends on he in e e ence wi h an igen p esen a ion;
lysosomal memb ane s abiliza ion, and inhibi ion he me abolism o deoxy ibonucleo ides (Fox
and Kang 1993, Webe and Le i z 2000). Hyd oxychlo oquine is a ely used as mono he apy o
RA; i s main clinical u ili y is in combina ion wi h o he DMARDs.
Sul asalazine
Sul asalazine is clea ed in he in es ine by bac e ial o ganisms in o 5-aminosalicylic acid
and sul apy idine. Sul apy idine seems o exe he he apeu ic ac ion a e in es inal
abso p ion. The mechanism o ac ion o sul asalazine is based on neu ophil unc ion inhibi ion,
immunoglobulin le els educ ion, and in e e ence wi h T-cell unc ion ia supp ession o NF-κB
ac i a ion (Ca lin, Dju sa e e al. 1992, Gadangi, Longake e al. 1996). The mos common
oxici ies a e gas o in es inal- ela ed (nausea, omi ing, dia hea, abdominal pain) and
hema ologic in na u e (neu openia, h ombocy openia), bu he d ug is gene ally conside ed
sa e and well- ole a ed (Symmons, Salmon e al. 1988).
Le lunomide
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Le lunomide is an immunomodula o y d ug, is a compe i i e inhibi o o dihyd oo o a e
dehyd ogenase, enzyme equi ed o he de no o syn hesis o py imidines, necessa ies o he
ac i a ion o lymphocy es. The e o e, blockade o he py imidine-syn hesis pa hway has
an ip oli e a i e e ec s. Le lunomide is gene ally well ole a ed; hepa o oxici y and
gas oin es inal in ole ance a e i s mos impo an ad e se eac ion (Olsen and S ein 2004). I is
mos commonly used as an al e na i e o me ho exa e-in ole an pa ien s.
O he an i heuma ic d ugs
Gold sal s, hei mechanism o ac ion include he educ ion o ci cula ing B cells, immune
complexes, heuma oid ac o , and immunoglobulin le els (Hi oha a, Nakanishi e al. 1999).
Long ea men wi h gold sal s could p esen oxic eac ions, including mucocu aneous
eac ions, p o einu ia, and cy openias.
Minocycline, om e acycline amily, in addi ion o hei an imic obial ac ion, i has an i-
in lamma o y and immunomodula o y p ope ies (O'Dell, Paulsen e al. 1999). Among i s side
e ec s, include au oimmune synd omes (se um sickness, polya h i is, d uginduced lupus, and
asculi is), headaches, GI in ole ance, and a g eying skin pigmen a ion.
Cyclospo ine, a calcineu in inhibi o demons a ed e icacy in he ea men o RA (
Sawi zke AD
2005). Howe e , gi en he a ailabili y o sa e DMARDs and biological agen s, i s ole is now
limi ed o a hi d-line agen a e ailu e o i s - and second-line d ugs.
Biological agen s
TNF- α an agonis
TNF-α, an in lamma o y cy okine, ha is impo an in he pa hogenesis o RA, binds o
wo ecep o s, he ype 1 TNF ecep o (p55) and he ype 2 TNF ecep o (p75), ha a e
exp essed on many ypes o cells. The biologic ac i i y o TNF-α can be a enua ed by soluble
TNF ecep o s. Pa ien s wi h RA ha e high concen a ions o TNF-α in he syno ial luid. TNF-α
is localized o he junc ion o he in lamma o y pannus and heal hy ca ilage, and high syno ial
luid TNF-α concen a ions a e associa ed wi h he e osion o bone (Olsen and S ein 2004).
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In liximab
In liximab is a chime ic molecule ha joins he Fc egion o human immunoglobulin G1
(IgG1) wi h he a iable egion o a mouse an ibody agains TNF-α (Ellio , Maini e al. 2008). I
binds o soluble and memb ane-bound TNF-α wi h high a ini y, impai ing he binding o TNF-α
o i s ecep o ; inducing a complemen and an ibody-dependen esponse agains cells ha
exp ess TNF-α. In liximab can be used as mono he apy, bu adminis a ion in liximab plus
me ho exa e is mo e e ec i e (Lipsky, an de Heijde e al. 2000).
E ane cep
E ane cep is a soluble molecule composed o wo ecombinan p75 TNF- ecep o p o eins used
oge he o o m a dime (Nes o o 2005). Each o hese molecules is linked o he Fc po ion o
human IgG1 o p o ide hem a longe hal -li e. E ane cep is indica ed in he ea men o
e ac o y RA o o he s DMARDs included me ho exa e.
Adalimumab
Adalimumab is a ecombinan human IgG1 monoclonal an ibody ha binds o human
TNF-α wi h high a ini y, bo h impai ing cy okine binding o i s ecep o s and lysing cells ha
exp ess TNF-a on hei su ace. I has a long ci cula ing hal -li e (app oxima ely 10–20 hou s) and
can be sel -adminis e ed. Adalimumab is indica ed in he ea men o e ac o y RA o o he s
DMARDs included me ho exa e.
IL-1 an agonis
Anakin a
IL-1 is a p o-in lamma o y cy okine p oduced by monocy es, mac ophages, and some
specialized cells in he syno ial lining, has in lamma o y e ec s ha include he induc ion o IL-
6 and COX-2. In pa ien s wi h heuma oid a h i is, le els o IL-1- ecep o an agonis in he
damaged join a e diminished. Anakin a is a ecombinan o m o human in e leukin- 1– ecep o
an agonis ha a ge s he ype I in e leukin-1 ecep o ha is exp essed in many issues.
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Anakin a in combina ion wi h me ho exa e a e indica ed in pa ien s who ha e no esponded
o me ho exa e mono he apy (Cohen, Hu d e al. 2002).
An i-CD20
Ri uximab
Ri uximab is a monoclonal an ibody di ec ed agains an an igen, CD20, on he su ace o
all B cells o he han s em cells and p e-B lymphocy es. I does no a ec plasma cells since hey
do no possess CD20 on hei cell su ace. I exe s i s e ec by binding o CD20 on B cells and
causing cell lysis by bo h complemen -dependen and an ibody-dependen cell media ed
cy o oxici y. I has e icacy in RA pa ien s who ha e ailed con en ional DMARDs and in RA
pa ien s who ha e ailed an i- TNF agen s (Edwa ds, Szczepanski e al. 2004, Cohen, Eme y e al.
2006).
An i-CD80/86
Aba acep
Aba acep is a usion p o ein linking he ex acellula domain o human cy o oxic T-
lymphocy e associa ed an igen 4 (CTLA-4) o he Fc po ion o human IgG1. I wo ks by
compe ing o he binding be ween CD28 on he T cell and CD80/86 on he an igen-p esen ing
cell. This is an impo an co-s imula o y signal ha is essen ial o T cell ac i a ion (Fan and Leong
2007). I is e ec i e in pa ien s who ha e an inadequa e esponse o ei he me ho exa e o one
o mo e TNF-inhibi o s (Geno ese, Becke e al. 2005).
IL-6 an agonis
Tocilizumab
Tocilizumab is a humanized an i-human IL-6 ecep o an ibody ha speci ically inhibi s
he biological ac i i y o IL-6 by compe i i ely inhibi ing he binding o IL-6 o he IL-6 ecep o
(Paul-Ple ze 2006). Tocilizumab mono he apy has been shown o be e ec i e in he ea men
o RA and may be an op ion o pa ien s ha ha e side e ec s wi h combina ion biologics and
DMARDs (Mima and Nishimo o 2008).
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3.1.1. Benzoic acid de i ed
Yoon e al. ha e shown an i-in lamma o y e ec s o gallic acid using ib oblas -like
syno iocy es (RA-FLS) cells isola ed om he syno ial issues o pa ien s wi h RA. They
demons a ed ha gallic acid could induce apop osis in RA-FSL h ough egula ion o apop osis
ela ed p o ein exp essions such as caspase-3, Bcl-2, Bax, p53 and pAk . As well, gallic acid was
able o educe p o-in lamma o y cy okines (IL-1β, IL-6), chemokines (CCL-2/MCP-1, CCL-7/MCP-
3), COX-2, and MMP-9 gene exp ession (Yoon, Chung e al. 2013). On he o he hand, ea men
wi h p o oca echuic acid signi ican ly educed he paw swelling in adju an -induced a h i is
(AIA) in a s. Addi ionally, hese ea men could decease lipid pe oxides (LPO) exp ession and
eac i e ee adicals ni ic oxide (NO) le els and inc ease an ioxidan enzyme exp ession, such
as, supe oxide dismu ase (SOD), ca alase and glu a hione (GSH) in li e . The an ia h i ic ac i i y
o p o oca echuic acid was ound o be equi alen o ha o diclo enac sodium (Lende,
Kshi saga e al. 2011).
3.1.2. Cinnamic acid de i ed
I is well know ha T cells a e in ol ed in ini ialling disease p ocess and, e en mo e,
eme ging e idence sugges s ha hese cells also pe pe ua e RA pa hogenesis a he la e s age
by sec e ing a ious media o s, such as ecep o ac i a o o NK-kB ligand (RANKL), IL-17 and
os eopon in, which in luence os eoclas s and FLS (Cho, Yoon e al. 2004, Xu, Nie e al. 2005).
Rosma inic acid has shown has apop o ic ac i i y owa d T cells om RA pa ien s and u he
e i ied a ge T cell subse s. CD3+CD25+ ac i a ed T-cell subse s om mos o he RA pa ien s
displayed signi ican ly highe apop osis a es han did he PBMCs and o al CD3+ T cells.
Fu he mo e, ac i a ed and e ec o CD4+ T cells, including CD4+CD25+ and CD4+CD45RO+ T
cells, had a endency o being mo e suscep ible o osma inic acid-induced apop osis han ha
o es ing and naï e T-cell subse s. Rosma inic acid was able o induce he elease o cy och ome
c om mi ochond ia and he blockage o mi ochond ial depola iza ion inhibi ed apop osis (Hu ,
Suh e al. 2007). On he o he hand, Hsu e al., ha e epo ed ha Rosma inic acid dec eased

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he numbe o mul inuclea ed os eoclas s in a dose-dependen manne using os eoclas -
p ecu so cell line RAW264.7 s ained wi h a a e- esis an acid phospha ase (TRAP). The
e ec s o his phenolic acid on os ecla ogenesis we e u he con i med by using p ima y mu ine
bone ma ow-de i ed mac ophages (BMM) s imula ed wi h RANKL. Rosma inic acid was able o
signi ican ly educed he c- os, nuclea ac o o ac i a ed T cells c1(NFATc1), TRAP, OSCAR, DC-
STAMP, and ß3 in eg in mRNA exp ession h ough inhibi ion o phospho yla ion o p38 and ERK
MAPKs and blocking he IkB deg ada ion and sup essing he subsequen nuclea ansloca ion
o NF-kB p65 (Hsu, Cheng e al. 2011). Simila ac i i y showed chlo ogenic acid who inhibi ed
RANKL- media ed os eoclas di e en ia ion by down- egula ion o phospho yla ion o p38, JNK
and ERK MAPKs and IkB deg ada ion. Chlo ogenic acid, also sup essed he mRNA exp ession o
NFATc1, TRAP and OSCAR in RANKL- ea ed BMMs (Kwak, Lee e al. 2013). Acco ding o he in
i o s udies, osma inic acid has been e i ied ha amelio a ed CIA, as mani es ed by educ ion
o syno i is and deple ion o COX-2 posi i e cells in a ec ed join s (Youn, Lee e al. 2003). The
immunomodula o y e ec o chlo ogenic acid, was desc ibed by Chauhan e al. in AIA in a s. In
his model, chlo ogenic acid was able o con ol he o al (CD3) and di e en ia ed (CD4 and CD8)
T cells coun and sup essed CD80/86 co-s imula o y molecule. Also his phenolic acid had he
abili y o diminish Th1 cy okines (IL-2, IFN-γ and IL-12) and ele a e Th2 cy okines (IL-4 and IL-10)
in CD4
+
cells (Chauhan, Sa i e al. 2012). p-cuma ic acid also showed immunomodula o y and
an i-in lamma o y in AIA in a s. The cinnamic acid de i ed imp o ed he a h i ic disease
h ough educed he mac ophage phagocy ic index, ci cula ing immune complex in se a and
syno ial TNF-α exp ession (P agasam, Venka esan e al. 2013).
3.2. Fla onoids
3.2.1. Fla ones
Apigenin and lu eolin a e la ones abundan ly p esen in common ui s and ege ables
such as chamomile ea and cele y among o he s ood. I has been demons a ed ha apigenin
has po en ial as an ia h i ic agen using in i o model (Shin, Kim e al. 2009). This die a y- plan
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la onoid was able o inhibi he p oli e a ion o RA-FSLs by inducing apop osis h ough
ac i a ion o he e ec o s caspase-3 and caspase-7. Apigenin ea men esul ed in ac i a ion o
ERK MAPK, and when he cells we e ea ed wi h he ERK inhibi o , apigenin-induced apop osis
was educed.
On he o he hand, he au ho s demons a ed ha he induc ion o he apop osis and
he ac i a ion o ERK by apigenin, i was due o he apigenin-induced gene a ion o ROS (Shin,
Kim e al. 2009). Simila esul s showed Sun e al., apigenin ea men was able o induce
apop osis o RA-FSLs, which was coupled wi h inc eased caspase-3 exp ession and ac i i y and
dec eased Bcl-2/Bax a io. These esul s we e likely associa ed wi h down egula ion o PI3-K/Ak
ac i i y (Sun, Jiang e al. 2012). Hou e al., epo ed ha lu eolin inhibi ed he p oli e a ion o
collagen-induced a h i is (CIA) a syno ial ib oblas s, which was accompanied by a dec ease
in MMP-1 and MMP-3 sec e ion. Besides, cy okine p oduc ion (IL-6, IL-8, IL-15 and TGF-β) we e
ound o be lowe in CIA a syno ial ib oblas s ea ed wi h lu eolin. The au ho s also
demons a ed ha lu eolin ea men caused a delay o cells in he G2/M phase and his
polyphenol inhibi ed he MAPK/ERKs and PI3K-Ak pa hways (Hou, Wu e al. 2009). O he in
i o s udy on SW982 cell line, demons a ed ha lu eolin ea men esul ed in inhibi ion o IL-
1β-induced MMP-1, MMP-3, TNF-α and IL-6 p oduc ion. Mo eo e , IL-1b-induced ac i a o
p o ein-1 (AP-1) and NF-κB ac i a ion we e inhibi ed by lu eolin (Choi and Lee 2010). Nobile in
is a ci us la one p esen especially in ange ines. Nobile in shown an i-in lamma o y e ec s
since i was able o down egula e he p oduc ion o MMP-1, MMP-3 and MMP-9 and PGE
2
in IL-
1 s imula ed abbi syno ial ib oblas , mo eo e his ci us polyphenol, inhibi ed he
p oli e a ion o hese cells (Ishiwa, Sa o e al. 2000). This ci us la one amelio a ed CIA in mice.
The educed a h i is index and se e i y we e accompanied by a supp ession o
os eoclas ogenesis and TRAP ac i i y in RAKNL-s imula ed RAW264.7 cells. Mo eo e , nobile in
ea men esul ed in inhibi ion o ac i a ion o MAPKs as well as AP-1/NF-kB pa hways
(Mu akami, Song e al. 2007). On he o he hand, in ano he CIA s udy, nobile in was able o
In oducción
22
imp o e he se e i y o damage and he au ho s, also demons a ed ha his la one educed
agg ecanase-1/a disin eg in and MMP wi h h ombospondin-like mo i s (ADAMTS)-4 and
ADAMTS-5 mRNA exp ession in CIA mice (Imada, Lin e al. 2008).
3.2.2. Fla onols
Que ce in, kaemp e ol and my ice in a e ound in se e al oods, including onions,
be ies, g apes and ed wine and exhibi a wide ange o an ia h i ic e ec s. Sa o e al.
demons a ed in 1997 ha que ce in sup esses IL-8 and MPC-1 mRNA in human syno ial
ib obla s s imula e wi h TNF-α. H
2
O
2
media ed induc ion o IL-8 and MCP-1 genes was also
inhibi ed by que ce in in syno ial ib obla s. Besides que ce in inhibi ed he induc ion o NF-kB
by TNF-α (Sa o, Miyazaki e al. 1997). O he s udy in RA-FLS ea ed wi h que ce in epo ed ha
que ce in inhibi ed he IL-1β-induced p oli e a ion. In addi ion, que ce in down egula ed he
exp ession o MMP-1, MMP-3, COX-2 and p oduc ion o PGE
2
in RA-SFs ia inhibi ion o IL-1β-
induced ac i a ion o NF-kB and phospho yla ion o p38, JNK and ERK MAPKs pa hway (Sung,
Lee e al. 2012). The apop o ic pa hway o que ce in in RA-SFs was desc ibed by Xiao e al., DNA
agmen a ion assay showed ha que ce in ele a ed he apop osis o RA-SFs, accompanying
wi h enhanced caspase-3 and caspase-9 clea ages. Mo eo e , que ce in caused loss o
mi ochond ial memb ane po en ial and cy och ome C elease o cy osol and also dec eased Bcl-
2/Bax a io. Addi ionally, que ce in ele a ed phospho yla ion a se 15. Indica ing ha que ce in-
induced apop osis o RA-SFs was h ough mi ochond ial pa hway, in which p53 played an
impo an ole (Xiao, Hao e al. 2013). In i o an ioxidan s s udies ha e e ealed he sca enging
ac i i y o ABTS and DPPH adicals, inhibi ion o ni ic oxide and he supe oxide adical
sca enging capaci y o que ce in (using hioglycolic acid-capped cadmium ellu ide quan um
do s (QDs) as nanoca ie ) (Jeyade i, Si asudha e al. 2013). Adminis a ion o QDs-que ce in
complex showed a educ ion in in lamma ion and imp o emen in ca ilage egene a ion in AIA
in a s. His ology o hind limb issue con i med he comple e ca ilage egene a ion in AIA a s
ea ed wi h QDs-que ce in complex. This ea men educed he exp essions lipid pe oxida ion
In oducción
23
and showed inc ease in ac i i ies o an ioxidan enzymes such as SOD, GSH, glu a hione
pe oxidase (GPx), ca alase (CAT) le els in paw issue. C- eac i e p o ein, heuma oid ac o , ed
blood cells and whi e blood cells coun and e y h ocy e sedimen a ion a e o expe imen al
animals we e also b ough back o no mal le els (Jeyade i, Si asudha e al. 2013). O al
adminis a ion o que ce in in a model o AIA amelio a ed all ma ke s o in lamma ion such as
MCP-1, IL-1β and C- eac i e p o ein and ma ke s o oxida i e s ess such as 13/15 LOX es o ing
plasma an ioxidan capaci y. A p o ec i e e ec o que ce in was e i ied being ha HO-1
p o ein le els we e up egula ed in he animals ea ed. In his s udy, he au ho s deepened
signalling pa hways possibly in ol ed, in his way, hey demons a ed ha que ce in was able
o inhibi phospho yla ion o ERK MAPKs and ac i a ion o NF-kB pa hway (Ga di, Baue o a e
al. 2015). On he o he hand, kaemp e ol has also shown exe an i-in lamma o y and an i-
a h i ic p op ie ies. As desc ibe Yoon e al., kaemp e ol inhibi s IL-1β- induced p oli e a ion o
RA-SFs by apop o ic way. Likewise, kaemp e ol ea men down egula ed he exp ession o
MMP-1, MMP-3, COX-2 and he p oduc ion o PGE
2
in IL-1β-s imula ed RA-SFs. Acco ding o he
in ol ed pa hways, his la onol inhibi ed he phospho yla ion o p38, JNK and ERK MAPKs and
he ac i a ion o NF-kB signalling pa hways (Yoon, Lee e al. 2013).
A ecen s udy es ing a possible immunomodula o y ac i i y o kaemp e ol in a h i is
showed ha kaemp e ol could s eng hen he supp ession unc ion o T eg cells and p e en
he pa hological symp om o CIA in a a model. Mechanis ically, kaemp e ol ea men could
s abilize FOXP3 p o ein le el in i o and in i o. Fu he mo e, his polyphenol could inhibi
PIM1-media ed FOXP3 phospho yla ion a S422 o p omo e FOXP3 ansc ip ional ac i i y (Lin,
Luo e al. 2015). Finally, Lee e al., demons a ed ha my ice in was able o dec eased IL-1β-
induced p oduc ion o IL-6 and MMP-1 in SW982 syo ial cells. Mo eo e , my ice in diminished
he phospho yla ion o p38 and JNK MAPKs (Lee and Choi 2010).
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24
3.2.3. Fla anones
Fla anones a e polyphenolic compounds highly and almos exclusi ely p esen in ci us.
Expe imen al obse a ion ha e shown ha hese compounds, including hespe idin and na ingin
p esen an i-in lamma o y and an ia h i ic p op ie ies. O ally adminis a ion o hespe idin o
CIA mice, esul in imp o emen o clinical sco es. Fu he mo e, hespe idin ea men was able
o imp o e his ological ea u es such as educed he damage o in e chond al join s and
sup essed he inc eases o in il a ion o in lamma o y cells and pannus o ma ion. This ci us
la onoid also, down egula ed TNF-α mRNA exp ession in lesions (Kawaguchi, Ma uyama e al.
2006). Simila ly esul s, ha e been demons a ed by Li e al., using a model o AIA in a s.
Hespe idin sup essed seconda y paw swelling and educed he polya h i is index o AIA a s.
These mac oscopic esul s we e companied by amelio a ion o join des uc ion and inhibi ion
o bone des uc ion, syno ial hype plasia and in lamma o y cells in il a ion. In addi ion,
hespe idin enhanced concana alin-A (ConA)-induced T-lymphocy e p oli e a ion and eco e ed
ConA-induced IL-2 p oduc ion by solenocy es in compa ison wi h AIA con ol g oup. P oduc ion
o in lamma o y cy okines such as IL-1, IL-6 and TNF-α we e also de e mina e in pe i oneal
mac ophages om ea ed a s and s imula ed wi h LPS. In his way, hespe idin was able o
inhibi he p oduc ion o hese p o-in lamma o y cy okines (Li, Li e al. 2008). O he s udy o
hespe idin in CIA model in a s e eals he an i-in lamma o y and an ioxidan e ec s o his
molecule. Hespe idin ea men sup essed he e olu ion o CIA a s and amelio a ed he
changes a his ological le el and was able o es o e damage al e a ion o a g ea ex en .
Adminis a ion o hespe idin showed a signi ican educ ion in neu ophil ac i a ion and
in il a ion in he join s, also dec eased TBARS concen a ion by inhibi ing lipid pe oxida ion in
he ca ilage issue. The an ioxidan enzymes GSH and SOD we e up egula ed by hespe idin, and
ca ilage ca alase ac i i y and ni i e con en we e lowe ed (Uma , Kuma e al. 2013).
Finally, he an i-in lamma o y and an ia h i ic e ec o na ingin we e shown using an
AIA a model. In his s udy, adminis a ion o na ingin sup essed he paw swelling in AIA a s

In oducción
25
and dec eased his opa hological changes o join issues. Mo eo e , na ingin supp essed he
p oduc ion o TNF-α, IL-1β and IL-6 in se um o a h i ic a s. TUNEL assay demons a ed ha
na ingin induced apop osis o AIA syno ial cells ia egula ion o he p o ein exp ession o Bcl-2
and Bax (Zhu, Wang e al. 2015).
3.2.4. Iso la ones
Soy bean iso la ones ha e been iden i ied as phy ochemical he apeu ic la onoids o
he ea men o RA. Genis ein and daidzein possess s uc u al simila i ies wi h selec i e
es ogen ecep o modula o s such as amoxi en and syn he ic iso la ones (Mohammad-Shahi,
Haida i e al. 2011). In i o s udies ha e e ealed he an i-in lamma o y and an ia h i ic
p op ie ies o genis ein in human syno ial ib oblas s. Genis ein inhibi ed ancho age-dependen
p oli e a ion o syno ial cells induced by IL-1β, TNF-α, o EGF, i ually inhibi ed S-phase en y,
caused cell cycle a es in G1 es ic ion poin and inhibi ed colony g ow h o heuma oid
syno ial cells induced by EGF. Genis ein also down egula ed MMP-2 and MMP-9 exp ession
induced by IL-1β o TNF-α (Zhang, Dong e al. 2012). Genis ein ea men dec eased he
sec e ion o IL-1β, IL-6, and IL-8 om TNF-α-s imula ed MH7A cells. Fu he mo e, genis ein
p e en ed TNF-α-induced NF-κB ansloca ion as well as phospho yla ion o IκB kinase-α/β and
IκBα, and also supp essed TNF-α-induced AMPK inhibi ion (Li, Li e al. 2014).
In i o s udies ha e also shown an ia h i ic e ec o iso la ones. One s udy epo ed
ha genis ein o ally ea men in CIA a s, dec eased p ima y paw in lamma ion. Die a y
genis ein exhibi ed a di e en pa e n o immunomodula ion, augmen ing lymphocy e
ac i a ion wi h inc eased IL-4 sec e ion, bu dec eased IFN-γ by cul u ed spleen lymphocy es
subjec ed o CIA in lamma ion (Wang, Zhang e al. 2008). T ea men wi h genis ein and daidzein
in CIA a s esul ed in no only a educ ion in disease symp oms bu also a delay in he onse o
symp oms. The ea hickness in ea ed a s was lowe ed. P e en ion o he issue damage and
join in lamma ion was also obse ed ollowing ea men wi h wo soy iso la ones. Genis ein
and daidzein we e able o educe TNF-α, IL-6, adiponec in and lep in se um concen a ions
In oducción
26
(Mohammad-Shahi, Haida i e al. 2011). Mohammad-Shahi demons a ed ha genis ein and
daidzein also played an impo an ole on he ex a-a icula complica ions in CIA model in a s.
In his model, bo h iso la ones we e able o es o e pa aoxonase and a yles e ase enzyme
ac i i y, which was dec eased in se um om CIA a s. Pa aoxonase low concen a ion is
associa ed wi h a lowe HDL- choles e ol and a highe le el o malondialdehyde and C- eac i e
p o ein. Genis ein and daidzein could also e e se he inc eased le els o malondialdehyde
esul ing om CIA (Mohammadshahi, Haida i e al. 2013).
3.2.5. An ocyanins
An hocyanin belongs o a subg oup o bioac i e la onoids esponsible o he blue,
pu ple, and ed colou o many ui s, lowe s, and ege ables such as g apes, pommeg ana e
and be ies. I has been shown o ha e p o ec i e e ec s in in i o and in i o a h i is models.
Mal idin, majo g ape an hocyanin, was able o imp o e AIA clinical sco e; bo h he apeu ic and
p e en i e o al p o ocols o mal idin we e e icien in educing a h i ic signs. Finally, he le el
o ni i es was dec eased ollowing bo h he apeu ic and p e en i e ea men wi h mal idin
om pe i oneal mac ophages. Thus con i ming he educ ion o mac ophages in lamma o y
s a e in i o in he p esence o mal idin (Decendi , Mamani-Ma suda e al. 2013). Delphinidin,
p esen s in c anbe ies and Conco d g apes, was ound o be a speci ic inhibi o o his one
ace yl ans e ase. Delphinidin ea men inhibi ed NF-kB unc ion by inhibi ing ace yla ion-
dependen nuclea ansloca ion o p65 in TNF-α-s imula ed human heuma oid a h i is
syno ial cell line (MH7A). Fu he mo e, his an ocyanin e icien ly supp essed TNF-α-induced
exp ession o IL-6, COX-2, and IL-1β in MH7A cells (Seong, Yoo e al. 2011). Cyanidin-3-glucoside
inhibi ed ecep o ac i a o o RANKL-media ed os eoclas and o ma ion and down egula ed
he exp ession o os eoclas di e en ia ion ma ke genes (Acp5, C sK, Osca , Tm7s 4, A p6 0d2,
and N a c1) P e ea men di e en ia ion wi h cyanidin-3-glucoside conside ably educed he
induc ion o ERK, JNK, and p38 MAPKs by RANKL in os eoclas p ecu so cells. Fu he mo e,
cyanidin-3-glucoside d ama ically inhibi ed he exp ession o c-Fos and N a c1, which a e
In oducción
27
impo an ansc ip ion ac o s o os eoclas di e en ia ion and ac i a ion. The o ma ion o
os eoclas s in cocul u e o bone ma ow cells and cal a ia-de i ed os eoblas s was also inhibi ed
by cyanidin-3-glucoside ea men (Pa k, Gu e al. 2015).
3.2.6. Fla anols
Tea polyphenols ha e ecei ed conside able public a en ion due o he posi i e
associa ion be ween ea consump ion and bene icial heal h e ec s. The pu a i e heal h bene i s
a ibu ed o g een ea (Camellia sinensis, Theaceace) is due o high concen a ions o
polyphenolic compounds known as ca echins, including epigalloca echin-galla e (EGCG),
epigalloca echin (EGC), epica echin-galla e (ECG), and epica echin (EC).
One s udy showed ha EGCG p e ea men inhibi ed bo h he cons i u i e and IL-1β-
induced chemokine MCP-1/CCL2 p oduc ion, egula ed upon ac i a ion, no mal T-cell exp essed
and sec e ed (RANTES/CCL5) p oduc ion, g ow h- egula ed oncogene (G o-α/CXCL1)
p oduc ion, and epi helial neu ophil-ac i a ing pep ide 78 (ENA-78/CXCL5) p oduc ion, and
MMP-2 ac i a ion by RA-SFs (Ahmed, Pakozdi e al. 2006). This was achie ed by EGCG ia
selec i e inhibi ion o he IL-1β-induced p o ein kinase Cδ and NF-κB pa hways (Ahmed, Pakozdi
e al. 2006). I was also shown, ha EGCG was e ec i e in inhibi ing IL-1β-induced MMP-1,
MMP-3, and MMP-13 in human endon ib oblas s (Co ps, Cu y e al. 2004). Syno ial ib oblas
IL-6 p oduc ion has been shown o inhibi bone o ma ion and o concomi an ly s imula e bone
eso p ion and pannus o ma ion (C ons ein 2007). O he s udy showed ha EGCG
p e ea men inhibi s IL-1β-induced IL-6 and ascula endo helial g ow h ac o syn hesis in RA-
SFs (Ahmed, Ma o e e al. 2008). Yun e al., showed ha EGCG ea men esul ed in inhibi ion
o TNFα-induced p oduc ion o MMP-1 and MMP-3 a he p o ein and mRNA le els in RA-SFs by
inhibi ing AP-1 DNA binding ac i i y (Yun, Yoo e al. 2008). Ahmed e al., demons a ed ha
caspase-3 ac i a ion by EGCG supp essed RA-SFs g ow h, and his e ec was mimicked by Ak
and NF-κB inhibi o s. Mcl-1 deg ada ion by EGCG sensi ized RA-SFs o TNFα- induced clea age
o poly ADP- ibose polyme ase p o ein and apop osis (Ahmed, Sil e man e al. 2009).
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28
EGCG-con aining g een ea ex ac in d inking wa e amelio a ed CIA in mice. The educed CIA
incidence and se e i y was e lec ed in a ma ked inhibi ion o he in lamma o y media o s COX-
2, IFN-γ, and TNF-α in a h i ic join s o g een ea- ed mice. Addi ionally, o al IgG and ype II
collagen-speci ic IgG le els we e ound o be lowe in se um and a h i ic join s o g een ea- ed
mice (Haqqi, An hony e al. 1999). Mo inobu e al., showed ha EGCG ea men educed bone
eso p ion as de e mined by a a e- esis an acid phospha ase posi i e mul inuclea ed cells,
bone eso p ion ac i i y, and os eoblas -speci ic gene exp ession o he ansc ip ion ac o
NFATc1. They also s udied he in i o e ec o EGCG in CIA mice, whe e adminis a ion o EGCG
inhibi ed in lamma ion (Mo inobu, Biao e al. 2008). Ano he s udy showed ha EGCG
amelio a ed a h i is and mac ophage in il a ion, and caused a educ ion in he amoun o
MCP-1/CCL2-syn hesizing os eoblas s in a CIA model (Lin, Chang e al. 2008). EGCG o ally
ea men amelio a ed clinical symp oms, educed his ological sco es in a h i ic mice and also
lowe ed se um ype-II collagen IgG2a an ibodies. EGCG signi ican ly supp essed T cell
p oli e a ion and ela i e equencies o CD4 T cells, CD8 T cells and B cell subse s including
ma ginal zone B cells, T1 and T2 ansi ional B cells, while inc easing he equency o CD4+
Foxp3+ egula o y T cells (T egs) and indoleamine-2,3-dioxygenase (IDO) exp ession by CD11b+
dend i ic cells (DC). Join homogena es om EGCG- ed mice exhibi ed inc eased le els o N -2
and HO-1 compa ed wi h con ol CIA mice (Min, Yan e al. 2015). In a-a icula injec ion o EGCG
in CIA mice, showed a signi ican educ ion in ca ilage deg ada ion in p ophylac ic- and
he apeu ic-g oups, by his omo phological sco ing o he a icula ca ilage (Na a ajan, Madhan
e al. 2015). Adminis a ion o EGCG o a adju an -induced a h i is esul ed in an inhibi ion o
IL-6 le els in he se um and join s o EGCG- ea ed animals. This s udy also showed ha EGCG
enhances he syn hesis o soluble gp130 p o ein, an endogenous inhibi o o IL-6 signalling and
ans-signalling. The inhibi ion o a h i is in EGCG- ea ed a s co ela ed o he educ ion in
MMP-2 ac i i y in he join s compa ed wi h he ac i i y le el in a h i ic a s (Ahmed, Ma o e
e al. 2008). G een ea ex ac adminis a ion in d inking wa e amelio a ed a adju an -
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35
Tabla 1. In i o s udies o die a y polyphenols in RA.
Polyphenol Sou ce
Expe imen al
Sys em
Mechanism/s o Ac ion
E icac
ious []
Re
Phenolic acids
Benzoic acid de i ed
Gallic Acid
Tea
RA-FSL
Gallic acid induces apop osis o RA FLS h ough
egula ion o apop osis ela ed p o ein exp essions
(caspase-3, Bcl-2, Bax, p53 and pAk ) and educes
he exp ession o p o-in lamma o y 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 e al.
2013)
Cinnamic acid de i ed
Rosma inic
Acid
Basil, osema y,
hyme and
peppe min
RA PBMCs
Mu ine bone
ma ow-de i ed
mac ophages
Rosma inic Acid induces he p e e en ial apop o ic
ac i i y o ac i a ed T cells and e ec o T cells ia
mi ochond ial pa hway.
Rosma inic acid inhibi s RANKL-media ed os eoclas
di e en ia ion by down- egula ion MAPKs and NF-kB
pa hway and supp essing NFATc1, TRAP and OSCAR
mRNA exp ession
50 µM
60 and
120
µM
(
Hu , Suh e
al. 2007)
(Hsu, Cheng
e al. 2011)
Chlo ogenic
acid
Peach, p unes and
g een co ee
Mu ine bone
ma ow-de i ed
mac ophages
Chlo ogenic acid inhibi s RANKL-media ed os eoclas
di e en ia ion by down- egula ion MAPKs and NF-kB
pa hway and supp essing NFATc1, TRAP and OSCAR
mRNA exp ession
28, 70
and
141
µM
(
Kwak, Lee
e al. 2013)
Fla onoids
Fla ones
Apigenin
Pa sley, cele y
and chamomile
ea
MH7A cells
RA-FSL
Apigenin induces apop osis h ough ac i a ion o he
e ec o s caspase-3 and caspase-7 and ac i a ion o
ERK MAPK.
Apigenin induces apop osis o RA-FSL h ough
inc ease caspase-3 exp ession and ac i i y and
dec ease Bcl2/Bax a io, media ed by
down egula ion o PI3-K/Ak ac i i y
100
μM
20 µM
(
Shin, Kim
e al. 2009)
(Sun, Jiang
e al. 2012)
Lu
eolin
Cele y, b occoli,
g een peppe ,
pa sley and
hyme.
Ra syno ial
ib oblas s
SW982
Lu eolin inhibi s he p oli e a ion o syno ial
ib oblas s in CIA a s. Dec eases he sec e ion o
MMP-1 and -3 and he exp ession o IL-6, IL-8, IL-15,
and TGF-β, h ough inhibi ion o MAPK/ERKs and
PI3K-Ak pa hways.
T ea men wi h lu eolin signi ican ly inhibi ed IL-1β-
induced MMPs (MMP-1 and -3) and cy okines (TNF-α
and IL-6) p oduc ion. Lu eolin also inhibi ed JNK and
p38 MAPKs ac i a ion and he ansc ip ion ac o s
AP-1 and NF-κB.
20 µM
1 and
10 µM
(
Hou, Wu
e al. 2009)
(Choi and
Lee 2010)

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36
Nobile in
Tange ine
Rabbi syno ial
ib oblas s and
a icula
chond ocy es.
RAW264.7
Nobile in down egula es he p oduc ion o MMP-1,
MMP-3 and MMP-9 and PGE
2
and COX-2 o IL--
s imula ed abbi syno ial cells and inhibi s
p oli e a ion o abbi syno ial ib oblas s in he
g ow h phase.
Nobile in supp esses RANKL-induced
os eoclas ogenesis in mac ophages h ough
inhibi ion o MAPKs and he esul an egula ion o
ansc ip ion ac o s (AP-1 and NFκB)
4, 8,
16, 32
µM
4, 20
and 50
µM
(
Ishiwa,
Sa o e al.
2000)
(Mu akami,
Song e al.
2007)
Fla onols
Que ce in
Onion
RA-FSL
RA-FSL
RA-FSL
Que ce in supp esses TNF-α induced IL-8 and MCP-1
mRNA. H
2
O
2
media ed induc ion o IL-8 and MCP-1
genes a e inhibi ed by que ce in. Que ce in inhibi es
he ac i a ion o NF-kappa B by TNF-α.
Que ce in inhibi s IL-1β- induced p oli e a ion o RA-
FSL and down egula es MMP-1, MMP-3, COX-2
exp ession and PGE2 p oduc ion. Th ough inhibi ion
o ERK, p38, JNK phospho yla ion and ac i a ion o
NF-kB by IL-β
Que ce in induces he apop osis o RA-FSL, h ough
enhances caspase-3 and caspase-9 clea ages and
h ough mi ochond ial pa hway.
50, 100
and
200
µM
100
µM
100,
200
and
300
µM
(
Sa o,
Miyazaki e
al. 1997)
(Sung, Lee
e al. 2012)
(Xiao, Hao
e al. 2013)
Kaemp e ol
Apples, g apes,
oma oes, g een
ea, po a oes,
onions, b occoli,
B ussels sp ou s,
squash,
cucumbe s,
le uce, g een
beans, peaches,
blackbe ies,
aspbe ies, and
spinach
RA-FSL Kaemp e ol inhibi es he p oli e a ion o IL-1β-
s imula ed RA-FSL, as well as he mRNA and p o ein
exp ession o MMP-1, MMP-3, COX-2 and PGE
2
induced by IL-1β. Th ough inhibi ion o ERK, p38, JNK
phospho yla ion and ac i a ion o NF-kB by IL-β.
100
µM
(
Yoon, Lee
e al. 2013)
M
y ice in
Vege ables, ui s,
nu s, be ies, ea
and ed wine.
SW982 My ice in dec eases IL-1β-induced p oduc ion o IL-6
and MMP-1 in syno ial cells. Mo eo e , my ice in
diminishes he phospho yla ion o
JNK and p38 MAPKs.
10 µM
(
Lee and
Choi 2010)
Iso la ones
Genis ein
Lupin, a a beans,
soybeans, kudzu,
and pso alen
RA-FSL
MH7A cells
Genis ein inhibi s p oli e a ion o RA-FSL induced by
IL-1β, TNF-α and EGF; also inhibi s MMP-2 and
MMP-9 exp ession induced by IL-1β o TNF-α .
Genis ein dec eases he sec e ion o IL-1β, IL-6, and
IL-8 om TNF-α-s imula ed MH7A cells. Genis ein
p e en ed TNF-α-induced NF-κB ansloca ion as
well as phospho yla ion o IκB kinase-α/β and IκBα,
and supp essed TNF-α-induced AMPK inhibi ion.
37 µM
5, 10
and 20
µM
(
Zhang,
Dong e al.
2012)
(Li, Li e al.
2014)
An ocyanins
Delphinidin
G apes
MH7A cells Delphinidin inhibi s TNF-α-induced NF-κB unc ion
and supp esses exp ession o IL-6, COX-2 and IL-1β.
10 and
30 µM
(
Seong, Yoo
e al. 2011)
Cyanidin
G
apes, bilbe y,
blackbe y,
Os eoclas
p ecu so cells
Cyanidin inhibi s os eoclas s o ma ion and
down egula es exp ession o os eoclas
100
µM
(
Pa k, Gu e
al. 2015)
In oducción
37
bluebe y, che y,
c anbe y
di e en ia ion ma ke genes, inhibi ing
phospho yla ion o MAPKs
Fla anols
EGCG
G een ea
RA-FSL
Human endon
ib oblas
RA-FSL
RA-FSL
RA-FSL
EGCG inhibi s IL-1β-induced MCP-1/CCL2,
RANTES/CCL5, G o-α/CXCL1, ENA-78/CXCL5
p oduc ion ans MMP-2 ac i a ion, ia inhibi ion o
p o ein kinase Cδ and NF-κB pa hway.
EGCG inhibi s IL-1β-induced MMP-1, MMP-3 and
MMP-13
EGCG inhibi s IL-1β induced IL-6 and VEGF.
EGCG inhibi s TNF-α-induced MMP-1 and MMP-3
exp ession by inhibi ing AP-1 DNA binding ac i i y.
EGCG inhibi s TNF-α induced Mcl-1 p o ein
exp ession. EGCG ac i es caspase 3 ac i i y,
media ed ia down- egula ion o he TNFα-induced
Ak and NF-κB pa hways
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 e
al. 2006)
(Co ps,
Cu y e al.
2004)
(Ahmed,
Ma o e e
al. 2008)
(Yun, Yoo
e al. 2008)
(Ahmed,
Sil e man
e al. 2009)
Dia ylhep anoids a
nd a ylalkanones
Ginge ol
Ginge (
Zingibe
o icinale)
RA-FSL Ginge ex ac inhibi s TNF-α-induced IL-8 and IL-6
(
Ribel
-
Madsen,
Ba els e
al. 2012)
Cu cumin
Cu cuma longa
Human
chond ocy es
Co-cul u e
canine
mesenchymal
s em cells and
p ima y
chond ocy es
RA-FSL
RA-FSL, MH7A
HIG-82 abbi
syno iocy es
Cu cumin elie es supp ession o collagen ype II,
β1-in eg in and caspase-3 ac i a ion.
Cu cumin enhances he p oduc ion o collagen ype
II, CSPGs, β1-in eg in and ac i a ing MAPKs signaling
and supp essing caspase-3 and COX-2.
Cu cumin induces apop osis in RA-FSL by inhibi ion
o Blc-2 and X-IAP exp ession, ac i a ion o caspase-3
and caspase-9 and deg ada ion o PARP. Also,
inhibi s COX-2 exp ession and PGE
2
p oduc ion.
Cu cumin blocks IL-1β and PMA-induced IL-6
exp ession, blocks PMA-induced VEGF exp ession,
blocks ac i a ion o NF-κB and ERK1/2 and induces
cell dea h h ough apop osis.
Cu cumin inhibi s in lamma o y p ocesses h ough
supp ession o collagenase and s omelysin
exp ession.
50 µM
5 µM
50, 75
and
100
µM
12.5,
25 and
50 µM
1 and
10 µM
(
Shakibaei,
Schulze-
Tanzil e al.
2005)
(Buh mann,
Mobashe i
e al. 2010)
(Pa k,
Moon e al.
2007)
(Kloesch,
Becke e
al. 2013)
(Jackson,
Higo e al.
2006)
S ilbenes
Res e a ol
G apes
RA-FSL
Res e a ol induces apop osis h ough caspase-3,
caspase-9, caspase-8 and PARP clea age,
100
µM
(
Byun, Song
e al. 2008)
In oducción
38
Table 2. In i o s udies o die a y polyphenols in RA.
Polyphenol Sou ce
Expe imen a
l Sys em Mechanism/s o Ac ion
E icacious
[] Re
Phenolic acids
Bezoic acid de i ed
P o oca echuic
acid
Onions,
mush oom.
AIA in a s P o oca echuic acid inhibi s a h i is index,
dec eases LPO and NO and inc eases
an ioxidan s, SOD, ca alase and GSH in li e .
50 and 100
mg/Kg o ally
(
Lende,
Kshi saga
e al. 2011)
Cinnamic acid de i ed
p
-
Couma ic Acid
Peanu s,
oma oes,
ca o s and
ga lic.
AIA in a s p-couma ic acid educes he se um ci cula ing
immune complex le els. Syno ial issue o
a h i ic a ea ed wi h p-couma ic acid showed
a educ ion in immunos aining o TNF-α.
100 mg/kg
i.p.
(
P agasam,
Venka esan
e al. 2013)
Chlo ogenic
Acid
Peach,
p unes and
g een co ee
AIA in a s Chlo ogenic acid con ols he o al (CD3) and
di e en ia ed (CD4 and CD8) T cells coun ,
supp esses CD80/86 and supp esses he Th1
cy okines bu ele a es Th2 cy okines.
2.5, 5, 10, 20
and
40mg/kg.
o ally
(
Chauhan,
Sa i e al.
2012)
Rosma ic acid
Basil,
osema y,
hyme and
peppe min
CIA in mice Rosma ic acid educes he a h i ic index,
his ological damage and COX-2 exp ession.
50 mg/Kg
i.p.
(
Youn, Lee
e al. 2003)
Fla onoids
Fla ones
MH7A cells
RA-FSL
RA-FLS
mi ochond ial cy och ome c elease and AIF
ansloca ion o he nucleus.
Res e a ol induces apop osis h ough ac i a ion o
caspase-3 and caspase-9, dis up s mi ochond ial
memb ane po en ials as a esul o dec eased Bcl-XL
exp ession, allowing cy och ome c om he
mi ochond ia in o he cy osol, in a si uin 1-
dependen manne .
Res e a ol a enua es IL-1β-induced p oduc ion o
MMP-1, MMP-3 and MMP-9. qRT-PCR showed a
signi ican educ ion in he ela i e abundance o he
ansc ip s o OPG and RANKL.
Res e a ol a enua es TNF-α-induced p oduc ion o
IL-1β and MMP-3 ia inhibi ion o PI3K-Ak signaling
pa hway in RA FLS.
100
µM
6.25,
12.5,
25 and
50 µM
100
µM
(Nakayama,
Yaguchi e
al. 2012)
(Gleh ,
B eisach e
al. 2013)
(Tian, Chen
e al. 2013)
O he die a y polyphenols
Oleocan hal
EVOO
ATDC-5
ATDC-5
Oleocan hal inhibi s LPS-induced NO p oduc ion and
iNOS exp ession
Oleocan hal supp esses LPS-induced IL-6 and MIP-1α
exp ession.
10 and
25 µM
15 µM
(
Iacono,
Gomez e
al. 2010)
(Sco ece,
Gomez e
al. 2012)
In oducción
39
Nobile in
Tange ine
CIA in mice
CIA in mice
Nobile in supp esses a h i ic index.
Nobile in imp o es a h i ic p og ession and join
his ology and exe s a chond op o ec i e ac ion
om he agg ecanase-media ed ca ilage
des uc ion acco ding o he inhibi ion o gene
exp ession and p oduc ion o ADAMTS-4 and -5
2 and 20
mg/Kg i.p.
15, 30 and
60 mg/Kg
i.p.
(
Mu akami,
Song e al.
2007)
(Imada, Lin
e al. 2008)
Fla onols
Que ce in
Onions
AIA in a s
AIA in a s
Que ce in imp o es a h i ic p og ession and
join his ology, dec eases lipid pe oxida ion and
inc eases an ioxidan s, SOD, ca alase, GSH and
GPx.
Que ce in educes MCP-1, IL-1β, C- eac i e
p o ein and 13/15 LOX. Up egula es HO-1
exp ession and inhibi s ERK phospho yla ion and
NF-κB ac i a ion.
0.2 and 0.4
mg/Kg in o
hioglycolica
cid-
cappedcadm
ium ellu ide
quan umdo
s. O ally
150 mg/Kg
O ally
(
Jeyade i,
Si asudha
e al. 2013)
(Ga di,
Baue o a
e al. 2015)
K
aemp e ol
Apples,
g apes,
oma oes,
g een ea,
po a oes,
onions,
b occoli,
B ussels
sp ou s,
squash,
cucumbe s,
le uce, g een
beans,
peaches,
blackbe ies,
aspbe ies,
and spinach
CIA in a s Kaemp e ol inhibi s PIM1-media ed FOXP3
phospho yla ion a S422 o p omo e FOXP3
ansc ip ional ac i i y.
100 mg/Kg
(
Lin, Luo e
al. 2015)
Fla anones
Hespe idin
O ange and
lemon
CIA in mice
AIA in a s
CIA in a s
Hespe idin imp o es clinical soco e, his ological
ea u es and down egula es TNF-α mRNA
exp ession.
Hespe idin educes polya h i is index and
his ological ea u es. Enhances CoA-induced T-
lymphocy e p oli e a ion and eco e s IL-2
p oduc ion in solenocy es; also, inhibi s IL-1, IL-6
and TNF-α le el in pe i oneal mac ophages om
ea ed a s.
Hespe idin imp o es clinical soco e, his ological
ea u es and dec ases TBARS concen a ion by
inhibi ion o lipid pe oxida ion in he ca ilage.
Up egula es GSH and SOD an ioxidan enzymes
and down egula es ca ilage ca alase ac i i y and
ni i e le el.
150 mg/Kg
O ally
40, 80 and
160 mg/Kg
In agas ica
lly
160 mg/Kg
O ally
(
Kawaguchi
,
Ma uyama
e al. 2006)
(Li, Li e al.
2008)
(Uma ,
Kuma e
al. 2013)
Na ingin
G apes
AIA in a s Na ingin supp esses paw swelling and
his opa hological changes o join issue.
Down egula es TNF-α, IL-1β and IL-6 se um le el
20 and 40
mg/Kg
O ally
(
Zhu, Wang
e al. 2015)
In oducción
40
and induces apop osis ia egula ion o Bcl-2 and
Bax p o ein exp ession.
Iso la ones
Genis ein
Lupin, a a
beans,
soybeans,
kudzu, and
pso alen
CIA in a s
CIA in a s
CIA in a s
Genis ein modula es immune esponses in CIA
model. Supp esses he sec e ion o IFN-γ and
augmen s he IL-4 p oduc ion.
Genis ein imp o es disease symp oms and
educes TNF-α, IL-6, adiponec in and lep in
se um concen a ion.
Genis ein es o es pa aoxonase and a yles e ase
ac i i y and dec eases MDA le els.
80 mg/Kg
O ally
20 mg/Kg
O ally
20 mg/Kg
O ally
(
Wang,
Zhang e al.
2008)
(Mohamma
d-Shahi,
Haida i e
al. 2011)
(Mohamma
dshahi,
Haida i e
al. 2013)
Dai
d
zein
Soybeans
CIA in a s
CIA in a s
Daidzein imp o es disease symp oms and
educes TNF-α, IL-6, adiponec in and lep in
se um concen a ion.
Daidzein es o es pa aoxonase and a yles e ase
ac i i y and dec eases MDA le els.
20 mg/Kg
O ally
20 mg/Kg
O ally
(
Mo
hamma
d-Shahi,
Haida i e
al. 2011)
(Mohamma
dshahi,
Haida i e
al. 2013)
An ocyanins
Mal idin
Red wine
AIA in a s Mal idin is e icien in educing a h i is sings and
dec eases ni i es le el om pe i oneal
mac ophages om ea ed a s.
25 mg/Kg
O ally
(
Decendi ,
Mamani-
Ma suda e
al. 2013)
Fla anols
EGCG
G een ea
CIA in mice
CIA in mice
CIA in a s
CIA in mice
CIA in mice
EGCG-con aining g een ea ex ac educes
disease symp oms, inhibi s COX-2, IFN-γ and TNF-
α le el in a h i ic join and dec eases an i-CII IgG
se um le el.
EGCG amelio a es a h i is du ing he disease
cou se and educes his ologic sco es o a h i is.
Also educes he numbe o os eoclas s in he
pannus o
a h i ic paw join .
EGCG educes he se e i y o CIA and alle ia es
join des uc ion. Mo eo e , diminishes he
numbe o CCL2-p oducing os eoblas s and
ec ui men o CD68+ mac ophages.
EGCG amelio a es clinical symp oms, his ological
sco es in a h i is, IL-1β, IL-6, TNF-α, IFN-γ, IL-10
and an i-CII IgG se um le el. Also sup esses T cell
p oli e a ion and inc eases N 2 and HO-1 le els.
EGCG educes his opa hological ca ilage
deg ada ion.
2 g/L o
g een ea
ex ac in
wa e , ad
libi um as
he sole
sou ce o
d inking
wa e .
20 mg/Kg
i.p.
20 mg/Kg
i.p.
10 mg/Kg
O ally
12 mg/Kg
In a-
a icula
injec ion
(
Haqqi,
An hony e
al. 1999)
(Mo inobu,
Biao e al.
2008)
(Lin, Chang
e al. 2008)
(Min, Yan
e al. 2015)
Min 201
(Na a ajan,
Madhan e
al. 2015)

In oducción
41
AIA in a s
AIA in a s
EGCG inhibi s IL-6 and MMP-2p oduc ion. EGCG
enhances he syn hesis o gp130 p o ein and
endogenous inhibi o o IL-6 signalling.
G een ea ex ac amelio a es a h i ic damage
ia inhibi ion o IL-17 and up egula ion o IL-10
se um le el.
100 mg/Kg
i.p.
8 and 12 g/L
g een ea
ex ac in
d inking
wa e
(Ahmed,
Ma o e e
al. 2008)
(Kim,
Rajaiah e
al. 2008)
Dia ylhep anoids and a ylalkanones
Ginge
Ginge
(Zingibe
o icinale)
CIA in a s Ginge amelio a es he clinical sco e, ca ilage
des uc ion and dec eases se um le els o IL-1β,
TNF-α, IL-2, IL-6 and an i-CII IgG.
50, 100 and
200 mg/Kg
i.p.
(
Fo
uda and
Be ika
2009)
Cu cumin
Cu cuma
longa
CIA in mice
AIA in a s
AIA in a s
Cu cumin a enua es a h i ic index and educes
BAFF, IFN-γ and IL-6 se a le els.
Cu cumin amelio a es symp oms o a h i is and
p ese es adiological al e a ions. Cu cumin
educes TNF-α, MDA, ni i es C- eac i e p o ein
and ci ullina ed pep ide an ibody se a le els.
Cu cumin imp o es his opa hological changes,
dec eases IL-1β and TNF-α syno ial and se a
le els and inhibi NF-κB pa hway.
50 mg/Kg
i.p.
10 and 30
mg/Kg
Cu cumin
loaded solid
lipid
nanopa icle
s. O ally
50 mg/Kg
i. . o o ally
(nano o mul
a ion)
(
Huang, Xu
e al. 2013)
(A o a,
Kuhad e al.
2015)
(Zheng, Sun
e al. 2015)
S ilbenes
Res e a ol
G apes
In aa icula
injec ion o
LPS in abbi s
CIA in mice
AIA in a s
Res e a ol dec eases ca ilage des uc ion and
loss o ma ix p o eoglycan con en in he
ca ilage.
Res e a ol a enua es clinical pa ame e s and
bone e osion; associa ed wi h ma kedly educed
se um le els o p o-in lamma o y cy okines and
collagen-speci ic IgG and wi h educed numbe s
o Th17 cells and he p oduc ion o IL-17 in DLN.
Res e a ol educes ca ilage degene a ion,
educes he p oduc ion o COX-2 and PGE
2
and
inhibi s he p oli e a ion o Con-A spleen cells
2.3 mg/Kg
In aa icula
injec ion
20 mg/Kg
i.p.
10 and 50
mg/Kg
In agas ica
lly
(
Elmali,
Baysal e al.
2007)
(Xuzhu,
Komai-
Koma e al.
2012)
(Chen, Lu
e al. 2014)
O he die a y polyphenols
Punicalagin
Pomeg ana e
AIA in a s Punicalagin diminishes disease de elopmen and
paw edema
10 and 50
mg/Kg
i.p.
(
Jean
-
Gilles, Li e
al. 2013)
Ellagic acid
Blackbe ies,
c anbe ies,
pecans,
pomeg ana e
s, aspbe ies,
s awbe ies,
walnu s,
wol be ies,
and g apes
CFA-induced
polya h i is in
a s
Ellagic acid educes join in lamma ion and
his opa hological changes; also supp esses TNF-α
and IL-1β p oduc ion.
0.13, 0.325
and 0.65%
Topically
(
Mo,
Panichayup
aka anan
e al. 2013)
In oducción
42
Oleu opein
EVOO
CIA in mice Oleu opein amelio a es clinical symp oms and
his ological sco es in a h i is; also dec eases p o-
in lamma o y cy okines and chemokine plasma
le els and MPo ac i i y.
40 mg/Kg
i.p.
(
Impellizze
i, Esposi o
e al. 2011)
Hyd oxy y osol
EVOO
CIA in a s Hyd oxy y osol dec eases paw edema and
his ological damage and educes COX-2 and iNOS
exp ession.
5 mg/Kg
O ally
(
Sil a,
Sepodes e
al. 2015)
JUSTIFICACIÓN Y OBJETIVOS
Jus i icación y Obje i os
45
Según la O ganización Mundial de la Salud, las en e medades eumá icas son la causa
más ecuen e de incapacidad, de o igen no men al, en el mundo. En España es as pa ologías
es án elacionadas con el 50.7% de las incapacidades labo ales y son la p incipal causa de bajas
labo ales pe manen es. Especial ele ancia iene la a i is euma oide (AR), po sus
epe cusiones sob e la calidad de ida de los pacien es y el de e io o p og esi o que p oduce
es a en e medad. En e el 20% y el 30% de los pacien es con AR se con ie en en incapaci ados
pe manen es. Demog á icamen e, la AR es la o ma más común de a i is in lama o ia y
a ec a ap oximadamen e al0.5 - 1% de la población mundial, con un impac o económico
compa able con el de la en e medad a e ial co ona ia. Es a pa ología puede apa ece en
cualquie e apa de la ida, sin emba go la p e alencia aumen a con la edad de al o ma que
el 80% de odos los pacien es inician la en e medad en e los 35 y 50 años de edad y es unas 3
eces mayo en muje es que en homb es (Gonzalez Ce nadas, Rod iguez-Rome o e al. 2014).
La AR es una en e medad au oinmunológica in lama o ia, c ónica y sis émica que se
ca ac e iza po una sino i is e osi a simé ica, en la cual el ejido de g anulación de o igen
sino ial (pannus), in ade y e osiona el ca ílago y el hueso de las a iculaciones dia odiales.
La en e medad se desa olla con ci as ele adas de la elocidad de sedimen ación e i oci a ia,
p o eína C eac i a, p esencia de inmunocomplejos y el ac o euma oide. Los pacien es con
AR p esen an a ec ación gene almen e de las a iculaciones dia odiales de o ma simé ica.
Dicha en e medad iene, en ocasiones, un componen e ex aa icula , a ec ando a di e sos
ó ganos y sis emas. El de e io o p og esi o, a icula o ex aa icula , acaba p o ocando que
es os pacien es p esen en de o midad, dolo , incapacidad uncional y disminución de la
expec a i a de ida (Fauci 2010).
La e iología de AR se desconoce aunque ac o es gené icos, in ecciosos (in ección i al,
e.g. Vi us Epsein-Ba , P o eus, ubeola,) ambien ales ( abaco, si uaciones de es és y
auma ismo, obesidad, die a) y ho monales pa ecen es a in oluc ados en ías elacionadas y
complejas (Suzuki and Yamamo o 2015). Se han iden i icado múl iples genes que con ibuyen
a la p edisposición y suscep ibilidad a la en e medad undamen almen e el gen HLA-DRB1 del
complejo mayo de his ocompa ibilidad, PADI4, PTPN22, CCR6 y FCRL3, en e o os
(Yamamo o, Okada e al. 2015).
Aunque la AR ha sido conside ada como una pa ología au oinmune, no exis e consenso
sob e la na u aleza del au oan ígeno(s) o ac o es ambien ales que inicia la des egulación de la
espues a inmune, mayo men e o ques ados po mac ó agos, células T egulado as (T eg), T
Capí ulo 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, Ma ina Sánchez-Hidalgo, Susana Sánchez-Fidalgo, Ma ina Apa icio-So o,
Isabel Villegas, Ca alina Ala cón-de-la-Las a
Depa amen o de Fa macología, Facul ad de Fa macia, Uni e sidad de Se illa, España
RESUMEN
An eceden es del ema: Aunque la pa ogenia de la a i is euma oide (AR) sigue sin conoce se
en su o alidad, ac ualmen e se ha desc i o la exis encia de una des egulación en el balance
Th1/Th2 y Th17/células T egulado as en pacien es con AR. En las a iculaciones a í icas, se
p oduce un inc emen o en la exp esión de ci ocinas p oin lama o ias, ales como TNF-α, IL-1β o
IL-17, que juegan un papel des acado en el desa ollo y p og esión de la en e medad. Es e
aumen o se a ibuye, al menos en pa e, a una des egulación en las u as de señalización celula
p o agonizadas po las MAP cinasas (MAPKs), JAK/STAT y el ac o nuclea NF-κB. Po o o lado,
el ac o nuclea N 2, es una pieza cla e en la ansc ipción de dis in as enzymas an ioxidan es
como la hemo oxigenasa 1 (HO-1), se ha demos ado que la ac i ación de la exp esión génica de
es a p o eína es capaz de modula la espues a in lama o ia. El hecho de que el abo daje
a macológico de la AR implique se e os e ec os secunda ios, ha gene ado en los úl imos años
un auge en el uso de o as es a egias e apéu icas, en e las que des aca la e apia nu icional.
Recien es es udios epidemiológicos han con i mado que el consumo de acei e de oli a i gen
ex a (AOVE), den o del con ex o de la die a Medi e ánea, esul a e icaz en la p e ención de
cie as pa ologías elacionadas con el es és oxida i o, la in lamación y el sis ema inmune. Po
o o lado, ecien es publicaciones de nues o g upo de in es igación, así como de o os au o es,
han demos ado las p opiedades an iin lama o ias e inmunomodulado as de la die a de AOVE
en dis in os modelos expe imen ales de in lamación.
Obje i os: En base a los an e io es an eceden es, nos plan eamos e alua la in luencia de una
die a de AOVE de la a iedad picual, en un modelo de a i is expe imen al inducida po
colágeno ipo II en a ones.
Ma e ial y Mé odos: Ra ones DBA-1/J ecién des e ados, ue on dis ibuidos en cua o g upos
expe imen ales: (1) Ra ones sanos alimen ados con die a de acei e de gi asol (CS-AG), (2)
Ra ones a í icos alimen ados con die a de acei e de gi asol (CIA-AG), (3) Ra ones sanos

Capí ulo I
54
alimen ados con die a de AOVE (CS-AOVE), (4) Ra ones a í icos alimen ados con die a de
AOVE. Después de seis semanas, la a i is ue inducida. El día 0, los a ones ue on inmunizados
con una inyección en la base de la cola de 100 mg de colágeno II. El día 21, ecibie on una
inyección de e ue zo in ape i oneal, con la misma can idad de colágeno. El g ado de desa ollo
de la en e medad ue alo ado de o ma isual siguiendo una escala de 0-2, dónde 0=sin
in lamación; 1=in lamación le e; 1.5=in lamación ma cada; 2=in lamación se e a y según el
es udio his ológico de las a iculaciones. Los ni eles en sue o de la p o eína oligomé ica de la
ma iz del ca ílago (COMP) y de me alop o easa 3 (MMP-3) así como los ni eles de ci ocinas
p oin lama o ias como TNF-α, IL-1β e IL-17 en el homogenado de pa a ue on de e minados
median e la écnica de ELISA. Los cambios en la exp esion p o eica de HO-1, N 2 y de las
p o eínas implicadas en las ías de señalización celula MAPKs, JAK/STAT y NF-κB ue on
es udiadas median e wes e n blo .
Resul ados: la die a de AOVE edujo de o ma signi ica i a el edema a icula y la des ucción
del ca ílago, p e iniendo el desa ollo de la AR. Los ni eles sé icos de COMP y MMP-3, los
encon amos disminuidos en aquellos animales alimen ados con la die a de AOVE. La exp esión
de las ci ocinas p oin lama o ias TNF-α, IL-1β e IL-17 ue meno el ejido a icula de los a ones
a í icos que ecibie on la die a de AOVE, en compa ación con los CIA-AG. Además la ac i ación
de las ías de señalización MAPKs, JAK/STAT y NF-κB ue inhibida po la die a de AOVE. De
acue do con la exp esión p o eica de N 2 y HO-1, es u o aumen ada en las a iculaciones de
los a ones pe enecien es al g upo CIA-AOVE.
Conclusión: Es os esul ados con i man, po p ime a ez, que el AOVE mues a un ele ado
e ec o p o ec o en la p e ención y la p og esión de la AR expe imen al, p obablemen e a a és
de la ac i ación de la ía N 2/HO-1, e inac i ación de las ías MAPKs, JAK/STAT y NF-κB, con los
que pod ía se conside ado como un alimen o uncional de p ime a magni ud den o de la
e apia nu icional de pacien es con AR, si u u os ensayos clínicos con i masen es os esul ados
expe imen ales.
1 3
Eu J Nu
DOI 10.1007/s00394-015-0850-0
ORIGINAL CONTRIBUTION
Die a y ex a‑ i gin oli e oil p e en s in lamma o y esponse
and ca ilage ma ix deg ada ion in mu ine collagen‑induced
a h i is
Ma ía Angeles Rosillo · Ma ina Sánchez‑Hidalgo ·
Susana Sánchez‑Fidalgo · Ma ina Apa icio‑So o ·
Isabel Villegas · Ca alina Ala cón‑de‑la‑Las a
Recei ed: 28 Augus 2014 / Accep ed: 13 No embe 2014
© Sp inge -Ve lag Be lin Heidelbe g 2015
o JAK/STAT, MAPKs and NF-κB pa hways was d as i-
cally amelio a ed. Acco ding o N 2 and HO-1, he p o-
ein exp essions we e up- egula ed in hose mice ed wi h
EVOO.
Conclusion These esul s suppo he in e es o EVOO as
a bene icial unc ional ood o p e en he de elopmen o
he heuma oid a h i is (RA).
Keywo ds CIA · EVOO · In lamma o y esponse · Oli e
oil · Rheuma oid a h i is
In oduc ion
Rheuma oid a h i is (RA) is an au oimmune disease cha -
ac e ized by ch onic in lamma ion o syno ial memb anes
and p oli e a ion o he syno ial lining leading o syno ial
hype plasia and massi e in il a ion o immune cells, as-
culogenesis, ca ilage and bone des uc ion and p og essi e
join damage [1]. Al hough he pa hogenesis o RA is no
ully unde s ood, gene ic and immunologic ac o s clea ly
con ibu e o he join damage du ing RA [2]. In ac , dys-
egula ed balances o Th1/Th2 and Th17/T eg cells ha e
been demons a ed in RA pa ien s [3].
RA syno ium is cha ac e ized by up- egula ed p oin-
lamma o y cy okines le els such as umo nec osis ac-
o alpha (TNF-α) and in e leukin (IL)-1β, he wo key
cy okines in ol ed in des uc i e a h i is in addi ion o
IL-17, IL-21, IL-23 and IL-6, among o he s. In ac , IL-17
plays impo an oles in he addi i e/syne gis ic e ec s
induced oge he wi h TNF-α and IL-1β, and ca ilage
ma ix b eakdown induc ion h ough he chond ocy e
me abolism dys egula ion. Besides, cy okines con ibu e
o he os eoclas s and mac ophages ac i a ion and also p o-
mo ion o ec ui men o bo h neu ophils and monocy es
Abs ac
Pu pose Cu en expe imen al s udies suppo a bene i-
cial ole o ex a- i gin oli e oil (EVOO) in se e al in lam-
ma o y diseases. The p esen s udy was designed o e alu-
a e he e ec s o die a y EVOO on ype II collagen-induced
a h i is (CIA) in mice.
Me hods DBA-1/J mice we e andomized in ou expe i-
men al g oups (10 o 15 animals pe g oup): (1) Sham sun-
lowe die (SO-Sham), (2) CIA sun lowe die (SO-CIA),
(3) Sham EVOO die (EVOO-Sham) and (4) CIA EVOO
die (EVOO–CIA) g oup. A e 6 weeks, a h i is was
induced by ype II collagen. Mice we e sac i ied 42 days
a e i s immuniza ion. In addi ion o mac oscopic and
his ological analyses, se um le els o ca ilage olime ic
ma ix p o ein (COMP), me allop o einase-3 (MMP-3)
and p o-in lamma o y cy okines le els we e e alua ed by
ELISA. The exp essions o heme oxygenase-1 (HO-1),
nuclea ac o E2- ela ed ac o 2 (N 2), mi ogen-ac i a ed
p o ein kinases (MAPKs), Janus kinase-signal ansduce
and ac i a o o ansc ip ion (JAK/STAT) and nuclea an-
sc ip ion ac o -kappa B (NF-κB) pa hways we e s udied
by wes e n blo ing.
Resul s EVOO die signi ican ly educed join edema and
ca ilage des uc ion, p e en ing he a h i is de elopmen .
Die a y EVOO signi ican ly dec eased se um COMP and
MMP-3 le els, as well as, he p o-in lamma o y cy okines
le els (TNF-α, IL-1β and IL-17). Mo eo e , he ac i a ion
M. A. Rosillo (*) · M. Sánchez-Hidalgo · S. Sánchez-Fidalgo ·
M. Apa icio-So o · I. Villegas · C. Ala cón-de-la-Las a
Depa men o Pha macology, Facul y o Pha macy, Uni e si y
o Se ille, P o eso Ga cía González, 2, 41012 Se ille, Spain
e-mail: [email p o ec ed]
C. Ala cón-de-la-Las a
e-mail: [email p o ec ed]
Eu J Nu
1 3
by means o inducing a ious chemokines. The in il a ed
leukocy es p oduce mo e cy okines and issue-des uc i e
enzymes leading o ch onic in lamma ion, ca ilage damage
and bone e osion [4].
The p ocess o gene exp ession o hese p o-in lam-
ma o y media o s in ol es mul iple signal ansduc ion
pa hways including mi ogen-ac i a ed p o ein kinases
(MAPKs) [5, 6] and nuclea ansc ip ion ac o -kappa
B (NF-κB) pa hways [6, 7]. Ac ually, NF-κB ac i a ion
esul s in me allop o einases (MMPs) p oduc ion, which
deg ade a icula ex acellula ma ix cons i uen s and
subsequen ly cause a icula des uc ion. In addi ion, he
Janus kinase–signal ansduce and ac i a o o ansc ip-
ion (JAK-STAT) is ano he ele an in lamma o y pa hway
ac i a ed in esponse o cy okines [8]. In pa icula , STAT-3
o e exp ession has been epo ed in syno ial memb anes
om RA pa ien s [9]. Likewise, nuclea ac o E2- ela ed
ac o 2 (N 2) is a key ansc ip ion ac o o ches a o o
he induc ion o se e al an ioxidan enzymes, such as heme
oxygenase (HO)-1. The ac i a ion o HO-1 gene exp es-
sion is conside ed o be an adap i e cellula esponse o
su i e exposu e o en i onmen al s esses. Mo e ecen ly,
HO-1 has also been epo ed o modula e in lamma o y
esponses, cell p oli e a ion and p e en apop osis [10, 11].
Recen esea ch has sugges ed ha die a y pa e ns, such
as he adi ional Medi e anean die , which is cha ac e -
ized by la ge amoun s o oods na u ally de i ed, in pa -
icula , ege ables, ui s, nu s, ish and g ains, may con e
p o ec ion om ce ain ch onic diseases ela ed o oxida-
i e s ess, in lamma ion and he immune sys em. In com-
pa ison wi h o he heal hy die s, Medi e anean die has a
high con en o o al a as i s mos dis inc i e ea u e due
o he usual high in ake o oli e oil om oli e ee, Olea
eu opaea [12].
Ex a- i gin oli e oil (EVOO) is ob ained om he ui
o he oli e ee solely by mechanical o o he physical
means unde condi ions ha do no lead o oil al e a ion.
T adi ionally, he bene icial e ec s o EVOO ha e been
a ibu ed o i s high MUFA con en (oleic acid) as i p o-
ec s lipop o eins and cellula memb anes, om oxida i e
damage [13]; howe e , nowadays, he e is inc easing e i-
dence o suppo he bene icial e ec s o EVOO in ake ha
a e due o i s mino highly bioac i e componen s (abou
1–2 % o oil weigh ) including phenolic compounds, i -
e pene alcohols, s e ols, hyd oca bons (squalene), i amins
(α- and γ- ocophe ols), β-ca o ene, phy os e ols, among
o he s [12, 14]. Pa icula ly, cu en expe imen al s udies
suppo a bene icial ole o polyphenols om EVOO in se -
e al in lamma o y diseases, including RA [15–18]. In ac ,
ou esea ch g oup has p e iously demons a ed ha an
EVOO die en iched wi h hyd oxy y osol, a phenolic com-
pound p esen in he oil, and a die a y EVOO-polyphenol
ex ac supplemen a ion could imp o e he in lamma o y
esponses associa ed wi h a ch onic expe imen al coli is
model [19, 20]. Fu he mo e, we also con i med bene icial
e ec s o die a y EVOO-unsaponi iable supplemen a ion
on an acu e expe imen al coli is model [21].
Al hough die a y EVOO has demons a ed immunomod-
ula o y and an iin lamma o y p ope ies in se e al in lam-
ma ion expe imen al models [22–24], ne e heless i s pos-
sible con ibu ion o RA emains unknown.
Taken his backg ound in o accoun , he p esen s udy
was designed o e alua e he po en ial p o ec i e e ec s o
die a y EVOO in collagen-induced a h i is (CIA) model,
a well-s udied animal model o RA use ul in he de el-
opmen o new he apies o RA [10], and o iden i y i s
unde lying molecula mechanisms. The se e i y o a h i-
is was assessed by mac oscopic and his ology pa am-
e e s. In lamma o y media o s and he signaling pa hways
in ol ed we e also explo ed.
Ma e ials and me hods
Animals and die s
A o al o 50 h ee-week-old male DBA-1/J mice (Jan ie ®,
Le Genes S Isle, F ance) we e main ained in ou Animal
Labo a o y Cen e unde s anda d condi ions ( empe a u e,
24–25 °C; humidi y, 70–75 %; ligh ing egimen, 12L/12D).
They we e ed pelle die s and wa e ad libi um. Mice we e
andomized in o ou expe imen al g oups (10 o 15 ani-
mals pe g oup) du ing all expe imen al pe iod: (1) Sham
sun lowe die (SO-Sham) g oup ecei ed a die elabo a ed
wi h a ma ke able sun lowe oil (Koipesol®, DEOLEO,
Spain), (2) CIA sun lowe die (SO-CIA) g oup ecei ed
a die elabo a ed wi h a ma ke able sun lowe oil (Koipe-
sol®, DEOLEO, Spain), (3) Sham EVOO die (EVOO-
Sham) g oup we e ed wi h a die made wi h a ma ke able
EVOO picual a ie y cha ac e ized by a high con en o
polyphenolic compounds (600 ppm) (Oleoes epa®, Se ille,
Spain) and (4) CIA EVOO die (EVOO-CIA) g oup we e
ed wi h a die made wi h a ma ke able EVOO picual a i-
e y, con aining high le els o polyphenolic compounds
(600 ppm) (Oleoes epa®, Se ille, Spain). All die s we e
o mula ed on he basis o he Ame ican Ins i u e o Nu i-
ion (AIN) s anda d e e ence die wi h he modi ica ion o
a ious sou ces o ca bohyd a e (Table 1), a 10 % o o al
oil (Table 2) and we e p epa ed by mixing he espec i e
compounds unde yellow ligh and s o ed a −80 °C. F esh
die s we e p o ided daily. The animals we e ed wi h he
co esponding die du ing 6 weeks p e ious o he CIA
induc ion and du ing he expe imen ime. Expe imen s ol-
lowed a p o ocol app o ed by he Animal E hics Commi -
ee o he Uni e si y o Se ille, and all expe imen s we e
in acco dance wi h he ecommenda ions o he Eu opean
Eu J Nu
1 3
Union ega ding animal expe imen a ion (Di ec i e o he
Eu opean Counsel 2010/630/EU).
Induc ion o CIA
A h i is was induced in 10-week-old male DBA-1/J mice.
Bo ine ype II collagen (CII) 2 mg/mL in dilu e ace ic acid
(MD Biop oduc s®, Zü ich, Swi ze land) was emulsi ied
in equal olumes o F eund’s comple e adju an (2 mg/
mL Mycobac e ium ube culosis, s ain H37Ra; Di co®,
De oi , Michigan, USA). On day 0, DBA-1/J mice we e
immunized a he base o he ail wi h 100 mg o bo ine
CII. On day 21, mice ecei ed an in ape i oneal boos e
injec ion o 100 mg o CII dissol ed in phospha e-bu e ed
saline (PBS). Mice we e conside ed o ha e a h i is when
signi ican changes in edness and/o swelling we e no ed
in he digi s o in o he pa s o he paws. Join in lam-
ma ion was sco ed isually in each paw, using a scale o
0–2 whe e 0 = unin lamed, 1 = mild, 1.5 = ma ked and
2 = se e e. Sco ing was pe o med by wo independen
obse e s wi hou knowledge o he expe imen al g oups.
Animals we e eu hanized on day 42 by o e doses o
pen oba bi al.
His ological analysis
A e mice we e killed, knee join s we e emo ed and
ixed in 4 % o malin. A e decalci ica ion in 10 % EDTA,
specimens we e p ocessed o pa a in embedding. Tissue
sec ions (7 mm) we e s ained wi h hema oxylin and eosin
(H&E) o pe o m his ological analysis.
Measu emen o in lamma o y ma ke s
Enzyme-linked immunoassay (ELISA) ki s we e used
o de e mine se um le els o ca ilage oligome ic ma ix
p o ein (COMP) le els, wi h sensi i i y o 0.2 U/L (MD
Biosciences®, Zu ich, Swi ze land) and ma ix me allo-
p o einase-3 (MMP-3) (sensi i i y o 10 pg/mL) (R&D
Sys ems®, Abingdon, UK). Hind paws we e ampu a ed
abo e he ankle and homogenized in 1 mL o 10 mM
Table 1 Composi ion o expe imen al die s (g/kg die )
Die was o mula ed on he basis o he AIN s anda d e e ence die
wi h he modi ica ion o a ious sou ces o ca bohyd a e
a Sun lowe oil om Koipesol® (Spain)
b EVOO om picual a ie y, Oleoes epa®, Se ille, Spain
c Mine al mix p o ided he ollowing (g/kg die ): calcium ca bon-
a e, 35.7; monopo assium phospha e, 25.0; sodium chlo ide, 7.4;
po assium sul a e, 4.66; po assium ci a e monohyd a e, 2.8; mag-
nesium oxide, 2.4; e ic ci a e, 0.606; zinc ca bona e, 0.165; man-
ganese ca bona e, 0.063; coppe ca bona e, 0.03; po assium ioda e,
0.001; sodium selena e, anhyd ous, 0.001025; ammonium molyb-
da e-4H2O, 0.000795; sodium me asilica e-9H2O, 0.145; ch omium
po assium sul a e-12H2O, 0.0275; bo ic acid, 0.00815; sodium luo-
ide, 0.00635; nickel ca bona e, 0.00318; li hium chlo ide, 0.00174;
ammonium anada e
d Vi amin mix p o ided he ollowing (g/kg die ): nico inic acid,
30 mg; D-calcium pan o hena e, 16 mg; py idoxine HCL, 7 mg; hia-
mine HCL, 6 mg; ibo la in, 6 mg; olic acid, 2 mg; D-bio in, 0.2 mg;
i amin B12, 25 mg; α- ocophe ol powde (250 U/g), 300 mg; i a-
min A palmi a e (250,000 U/mg), 16 mg; i amin D3 (400,000 U/g),
2.5 mg; phylloquinone, 0.75 mg
Ing edien s SO die EVOO die
Casein 200 200
DL-me hionine 3 3
Co n s a ch 150 150
Suc ose 447.3 447.3
Cellulose 50 50
Sun lowe oila100 –
EVOOb– 100
Mine al mixc35 35
Vi amin mixd10 10
Choline bi a a e 2 2
Table 2 Fa y acid, s e ols, squalene, i e pene alcohols and ocoph-
e ol composi ion o EVOO and SO
Pa ame e s EVOO SO
Fa y acid composi ion as de e mined by GC (% m/m me hyl es e s)
My is ic C-14:0 0.01 0.08
Palmi ic C-16:0 11.00 6.62
Palmi oleic C-16:1 0.90 0.12
Ma ga ic C-17:0 0.10 0.04
Ma ga oleic C-17:1 0.10 0.03
S ea ic 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
A aquic C-20:0 0.40 0.27
Eicosenoic C-20:1 0.20 0.22
Behenic C-22:0 0.10 0.58
Lignoce ic C-24:0 0.10 0.17
S e ols as de e mined by GC, ela i e amoun s (%)
Choles e ol 0.10 0.20
B assicas e ol 0.00 0.10
Campes e ol 3.30 10.70
S igmas e ol 0.50 8.80
β-si os e ol 95.40 63.40
Δ7-S igmas enol 0.40 10.50
Δ5-A enas e ol 0.00 6.2
To al s e ols (mg/kg) 1,351 3,832
Squalene as de e mined by GC (mg/kg) 5,772 –
T i e pene alcohols (mg/kg) 542 –
Tocophe ols and oco ienols as de e mined by HPLC (mg/kg)
α-Tocophe ol (Vi amin E) 85 575
Eu J Nu
1 3
4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid
(HEPES) bu e , pH 7.4. Supe na an s we e used o he
de e mina ion o IL-17, TNF-α and IL-1β le els, using
he ELISA ki wi h sensi i i ies o 4.6 (Diaclone®, Besan-
con Cedex, F ance), 25 (Diaclone®, Besancon Cedex,
F ance) and 5.1 (R&D Sys ems®, Abingdon, UK) pg/mL,
espec i ely.
Isola ion o cy oplasmic and nuclea p o eins
and immunoblo ing de ec ion
F ozen hind paws we e homogenized in liquid N2. Isola-
ion o cy oplasmic and nuclea p o eins was pe o med as
desc ibed by Rosillo e al. [25] wi h some modi ica ions.
P o ein concen a ion o he homogena e was de e mined
ollowing B ad o d’s colo ime ic me hod [26]. Aliquo s o
supe na an ha con ains equal amoun o p o ein (50 µg)
we e e alua ed o de e mine HO-1 (Enzo®, Mad id, Spain),
N 2, p-p38, p-JNK, p-STAT3, p65 (San a C uz Bio ec-
nology®, CA, USA) and IκB-α (Cell Signalling Technol-
ogy®, MA, USA) p o eins by Wes e n blo as desc ibed by
Rosillo e al. [25]. The signals we e analyzed and quan i-
ied by a scien i ic imaging sys ems (ImageJ so wa e®,
Ma yland, USA).
Da a analysis
All alues in he igu es and ex a e exp essed as a i hme-
ic mean ± s anda d e o (SEM). Da a we e e alua ed wi h
G aph Pad P ism e sion 5.04 so wa e. The s a is ical sig-
ni icance was e alua ed by wo-way ( ime cou se) o one-
way ( es o pa ame e s) analysis o a iance (ANOVA),
ollowed by Newman–Keuls’ es . P alues < 0.05 we e
conside ed s a is ically signi ican .
Resul s
E ec s o die a y EVOO on CIA model
The de elopmen o a h i is was moni o ed un il day 42.
The ime cou se o a h i ic sco e (Fig. 1a) indica es ha
con ol mice ed wi h SO p esen ed a p og essi e de el-
opmen o a h i is obse ed om day 29. Howe e , mice
eeding wi h EVOO showed a signi ican (p < 0.05 s. SO-
CIA) delayed onse and dec eased he disease se e i y o
CIA educing disease incidence, numbe o in ol ed paws,
oo pad hickness and clinical index om days 30 o 42.
These esul s sugges ed ha die a y EVOO no only could
e a d he de elopmen bu i also may ha e a he apeu ic
e ec on ongoing in lamma o y a h i is. Rep esen a i e
pho og aphs o hind paws om he di e en expe imen al
animal g oups a e shown in Fig. 1b.
In addi ion, H&E s aining e ealed ha his ological ea-
u es o he join om sham animals we e ypical o no mal
s uc u e wi h syno ial memb ane composed o syno ial
cells and collagen and a clea syno ial space (Fig. 2a,b). On
he con a y, join om SO-CIA mice exhibi ed his ologi-
cal changes indica i e o se e e a h i is, cha ac e ized by
an ex ensi e in il a ion o in lamma o y cells in o a icula
issues, exuda ion in o he syno ial space, syno ial hype -
plasia and ca ilage e osion (Fig. 2c). These his ological
ea u es we e less e iden in EVOO-CIA g oup (Fig. 2d).
E ec s o die a y EVOO on se um bioma ke s
MMP-3 is well known as a p edic o o join des uc ion
in ea ly o es ablished RA. In he p esen s udy, we de e -
mined se um MMP-3 le els, as syno ial in lamma o y bio-
ma ke , in CIA mice o in es iga e he po en ial bene icial
e ec s o ou nu i ional he apeu ic s a egy. Ci cula ing
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
A i ic Sco e
A
B
cd
ba
Fig. 1 Time cou se o he a h i is mac oscopic sco e (a), ep esen a-
i e pho og aphs (b) o hind paws a he end o he expe imen (day
42). SO-Sham (a), non-a h i ic mice eed wi h SO die ; EVOO-Sham
(b), non-a h i ic mice eed wi h EVOO die ; SO-CIA (c), con ol
a h i ic g oup eed wi h SO die ; EVOO-CIA (d), a h i ic mice eed
wi h EVOO die . Da a ep esen mean ± SEM n = 10. *p < 0.05 and
**p < 0.01 s Sham (SO and EVOO); +p < 0.05 s SO-CIA

Eu J Nu
1 3
MMP-3 le el was ma kedly inc eased (p < 0.001 s.
Sham) in se um om CIA animal g oup ed wi h SO die
(Fig. 3). By con as , a s ong educ ion in MMP-3 se um
le els was obse ed in hose a h i ic animals eeding wi h
EVOO (p < 0.001 s. SO-CIA) eaching le els compa able
o hose desc ibed in sham animals. In addi ion, se um le -
els o he ca ilage deg ada ion ma ke , COMP, we e sig-
ni ican ly ele a ed in SO-CIA animals when compa ed o
sham con ols. On he con a y, die a y EVOO signi ican ly
dec eased se um COMP le els in CIA mice in compa i-
son wi h hose SO-CIA animals (p < 0.001 s. SO-CIA)
(Fig. 3).
E ec s o die a y EVOO on join media o s
I has been epo ed ha TNF-α, IL-1β and IL-17 a e c i i-
cal cy okines in ol ed in he pa hogenesis o RA [27].
To explo e whe he cy okines join le els we e pa alleled
o he disease se e i y o CIA, concen a ion o hese
cy okines was examined in paw homogena es by ELISA.
As shown in Fig. 4, TNF-α, IL-1β and IL-17 le els we e
signi ican ly inc eased in paw homogena es om SO-CIA
animals when compa ed wi h sham mice (p < 0.001 and
p < 0.5 s. Sham), sugges ing i s ela ionship wi h he syn-
o ial issue in lamma ion. Con e sely, ou esul s indica e
ha animals ed wi h EVOO die showed a signi ican
educ ion in all hose p o-in lamma o y cy okines p oduc-
ion in compa ison wi h SO-CIA g oup (TNF-α: p < 0,01,
IL-1β: p < 0.001 and IL-17: p < 0.05 s. SO-CIA). Impo -
an ly, IL-1β and IL-17 cy okines alues we e simila o
hose ob ained om con ol sham animals.
E ec o die a y EVOO on p-STAT3 p o ein exp ession
STAT-3 has been desc ibed as a c i ical ansc ip ion ac-
o in ol ed in he pa hogenesis o RA by s ee ing he
abno mal ac i a ion, au oma ici y and p olonged su i al
o syno ial cells. Besides, STAT-3 is a key ansc ip ion
ac o in ol ed in Th17 cell di e en ia ion [28]. We e alu-
a ed p-STAT3 p o ein exp ession by Wes e n blo om
hind paw homogena es. S a is ical analysis e ealed a sig-
ni ican p-STAT-3 o e exp ession in SO-CIA g oup when
compa ed o he sham con ol g oups (p < 0.01 s. Sham),
whe eas nu i ional he apy wi h EVOO signi ican ly sup-
p essed STAT3 phospho yla ion in a h i ic CIA mice
(p < 0.01 s. SO-CIA) (Fig. 5). These esul s a e in acco d-
ance wi h hose ob ained in he measu emen o p o-in lam-
ma o y cy okines le els sugges ing ha die a y EVOO may
ep ess IL-17 p oduc ion ia nega i ely in e e ing wi h
STAT3 signaling pa hway.
Fig. 2 His ological analysis o he on al sec ions o knee join s on
day 42. Sec ions we e s ained wi h hema oxylin and eosin. O igi-
nal magni ica ion x200. SO-Sham (a), non-a h i ic mice eed wi h
SO die ; EVOO-Sham (b), non-a h i ic mice eed wi h EVOO die ;
SO-CIA (c), con ol a h i ic g oup eed wi h SO die ; EVOO-CIA.
A ows he in il a ion o in lamma o y cells and he ca ilage e osion.
d A h i ic mice eed wi h EVOO die . The a ow less damage a he
ca ilage. The images a e ep esen a i e o a leas six expe imen s
Eu J Nu
1 3
E ec o die a y EVOO on HO-1 and N 2 p o ein
exp ession
The exp ession o he p o eins HO-1 and N 2 was also
e alua ed in paw homogena es by wes e n blo ing. Ou
da a show ha HO-1 was signi ican ly down egula ed in
SO-CIA mice du ing he main enance o ch onic in lam-
ma ion (p < 0.001 s. Sham); howe e , die a y EVOO ea -
men induced a s ong HO-1 o e exp ession in compa ison
wi h SO-CIA a h i ic con ol g oup (p < 0.01 s. SO-CIA)
(Fig. 6). N 2 ac i a ion has been epo ed o play an
impo an ole in HO-1 exp ession. Acco ding o ou esul s,
nuclea N 2 exp ession was educed in hose a h i ic ani-
mals eed wi h SO die (p < 0.05 s. Sham), whe eas a sig-
ni ican inc ease in nuclea N 2 p o ein le els was obse ed
in EVOO-CIA animals (p < 0.05 s. SO-CIA) (Fig. 6).
E ec o die a y EVOO on JNK and p38 MAPKs
phospho yla ion
MAPKs play a key ole inducing he p o-in lamma o y
gene exp ession, which ini ia es in lamma o y esponses.
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 Measu emen o se um COMP and MMP-3 le els. These we e measu ed by ELISA ki s. Da a ep esen mean ± SEM, n = 10.
**p < 0.01 and ***p < 0.001 s Sham (SO and EVOO); +++p < 0.001 s SO-CIA
Sham CIA Sham CIA
0
100
200
300
*
+
SO EVOO
IL-17 (pg/g issue
)
Sham CIA ShamCIA
0
100
200
300
***
++
SO EVOO
TNF-α (pg/g issue)
Sham CIA Sham CIA
0
500
1000
1500
***
+++
SO EVOO
IL-1β (pg/g issue)
Fig. 4 Measu emen o TNF-α, IL-1β and IL-17 le els in hind paw homogena es. These we e de e mined by ELISA ki s. Da a ep esen
mean ± SEM, n = 10. *p < 0.05 and ***p < 0.001 s Sham (SO and EVOO); +p < 0.05, ++p < 0.01 and +++p < 0.001 s SO-CIA
Eu J Nu
1 3
We in es iga ed he e ec o die a y EVOO on MAPKs
(JNK and p38) signaling pa hway ac i a ion in CIA mice.
In he p esen s udy, phospho yla ion o JNK and p38 p o-
eins inc eased in cy osolic ex ac s om SO-CIA mice
paw homogena es. None heless, he p o ein exp ession
o bo h p-JNK and p-p38 was signi ican ly amelio a ed
in EVOO-CIA mice (p < 0.05 and p < 0.01 s. CIA-SO)
(Fig. 7).
E ec s o die a y EVOO on NF-κB signaling pa hway
We also in es iga ed he e ec s o EVOO on IkB-α deg a-
da ion in cy oplasmic ex ac s om mice join s. As shown
in Fig. 8, IκB-α exp ession was signi ican ly educed in
CIA-induced a h i is mice when compa ed wi h sham
mice, in he SO ea men . The IκB-α exp ession obse ed
in CIA mice is consis en wi h an inc ease in IκB-α deg a-
da ion, hus allowing NF-κB ansloca ion in o he nucleus
o bind speci ic DNA sequences leading o p o-in lam-
ma o y genes ansc ip ion. On he con a y, he e ec
obse ed in a h i ic con ol g oup on IκB-α p o ein exp es-
sion was p e en ed by die a y EVOO ea men . Acco d-
ing o he esul s ob ained, he nuclea p65 p o ein le els
we e inc eased in SO-CIA g oup (p < 0.001 s. Sham),
whe eas die a y EVOO ea men p e en ed he CIA-
induced nuclea ansloca ion le el o p65 in paw homoge-
na e in compa ison wi h hose a h i ic animals eed wi h
SO (p < 0.01 s. SO-CIA) (Fig. 8) a oiding he NF-κB-
media ed ansc ip ional ac i a ion.
Discussion
The ocus o his s udy has been o demons a e he po en-
ial p o ec i e ole o a die elabo a ed wi h EVOO in he
p e en ion and de elopmen o in lamma o y a h i is and
join damage in a mu ine CIA-induced expe imen al a h i-
is model. This model is commonly used o in es iga e
mechanisms ele an o RA as well as new an ia h i ic
ea men s [10]. CIA induc ion esul ed in he de elopmen
o a p onounced syno i is associa ed wi h an au oimmune
esponse agains ca ilage and p oduc ion o ma ix-deg ad-
ing enzymes accompanying ca ilage deg ada ion and bone
e osions [29]. The esul s o he p esen wo k clea ly indi-
ca e, o he i s ime, ha EVOO, as he lipid componen
o he die , e ec i ely exhibi ed p e en i e and he apeu-
ic e ec s in he de elopmen o in lamma o y a h i is
and join damage in CIA a h i ic mice in compa ison wi h
hose CIA mice ed wi h SO. This e ec was well co e-
la ed o an imp o ed a h i is sco e, a mino in lamma o y
cells in il a ion in o a icula issues, educed exuda ion
in o he syno ial space, syno ial hype plasia and ca ilage
e osion.
The pa hogenesis o RA is closely ela ed o an imbal-
ance o cy okine ne wo k con ibu ing o he de elopmen
and p og ession o bo h CIA and RA [30]. Ac ually, o e -
exp ession o p o-in lamma o y cy okines, such as IL-1β,
Sham CIA Sham CIA
0.0
0.5
1.0
1.5
***
++
SO EVOO
Densi ome y Analysis
(A bi a y uni s)
Sham CIA Sham CIA
SO EVOO
Fig. 5 Changes in p-STAT3 p o ein exp ession in hind paws
homogena e. The exp ession o p-STAT3 was quan i ied by den-
si ome y and no malized wi h espec o β-ac in. Da a ep e-
sen mean ± SEM, n = 4.***p < 0.001 s Sham (SO and EVOO);
++p < 0.01 s SO-CIA
Sham CIA Sham CIA
0
1
2
3
4N 2 HO-1
SO EVOO
***
*
++
+
Densi ome y Analysis
(A bi a y uni s
)
Sham CIA Sham CIA
SO EVOO
Fig. 6 EVOO die a y up- egula ed HO-1 and N 2 p o ein exp es-
sion in hind paws homogena e. The exp ession was quan i ied by
densi ome y and no malized wi h espec o β-ac in. Da a ep esen
mean ± SEM, n = 4.*p < 0.05 and ***p < 0.001 s Sham (SO and
EVOO); +p < 0.05 and ++p < 0.01 s SO-CIA
Eu J Nu
1 3
TNF-α and IL-17, may ac i a e os eoclas s and mac-
ophages and also ec ui leukocy es in in lamed join s.
The in il a ed leukocy es p oduce cy okines, chemokines
and issue-des uc i e enzymes leading o ch onic in lam-
ma ion, ca ilage damage and bone e osion. Besides, IL-17
is able o induce he elease o IL-8 and IL-6, and plays a
ema kable ole in he addi i e/syne gis ic e ec s induced
by TNF-α and IL-1β [31]. Impo an ly, a ge ing IL-17
has demons a ed he apeu ic e ec s in animal models o
in lamma o y and au oimmune diseases, including RA [32].
Cy okines also up- egula e MMPs exp ession by chond o-
cy es and syno iocy es a he pannus ca ilage junc ion
leading o ca ilage deg ada ion [3, 4, 33]. The e o e, we
examined TNF-α, IL-1β and IL-17 le els in paw homoge-
na es om CIA animals in compa ison wi h sham con ol
g oup. Ou esul s indica e ha animals ed wi h EVOO
die showed a signi ican educ ion in all p o-in lamma o y
cy okines le els. Consequen ly, EVOO in ake would con-
ibu e o educe he in lamma o y esponse and join dam-
age by supp essing he p oduc ion o he key Th17 pola i-
za ion cy okines, TNF- α and IL-1β, in CIA a h i ic mice.
COMP is a ma ix p o ein wi h a g ea po en ial as
a biological ma ke o ca ilage me abolism in a h i is
[34]. Inc eased agmen s o COMP ha e been epo ed in
pa ien s wi h os eoa h i is, RA and join inju y; hus, he
moni o ing o COMP le els in syno ial luid o se um has
been sugges ed o be a help ul me hod o assessing he
p esence and p og ession o he in lamma o y disease. In
his sense, se um COMP le els a e educed in RA pa ien s
in emission [34, 35]. In addi ion, COMP is a pu a i e
subs a e o MMPs. Pa icula ly, MMP-3 is a p o einase
sec e ed by syno ial ib oblas s and chond ocy es. I s ac i -
i y esul s in deg ada ion o agg ecan co e p o ein, ca ilage
link p o ein, ib onec in and collagen ypes IV, VII, IX and
XI. MMP-3 is p esen in RA syno ial luid, o e exp essed
in heuma oid syno ium and hen associa ed wi h highe
join damage in RA [36]. Likewise, se um MMP-3 le el is
sugges ed as a p edic o o join des uc ion in ea ly RA
o es ablished RA and should be used in associa ion wi h
usual in lamma o y ma ke s o ollow he apy e iciency
[37]. Ou da a a e in ag eemen wi h abo e s udies and
showed ha high se um COMP and MMP-3 le els we e
associa ed wi h disease ac i i y and join p og ession in
CIA mice; by con as , he p oduc ion o bo h ca ilage and
syno ial bioma ke s was signi ican ly inhibi ed by die a y
EVOO ea men in CIA mice.
Abno mal signaling pa hways play an impo an ole
in he in lamma o y p ocess and can lead o dys egula-
ion o he in lamma o y esponse being c ucial in RA
Sham CIA ShamCIA
0.0
0.5
1.0
1.5 p-p38 p-JNK
*** **
++
+
SO EVOO
Densi ome y Analysi
s
(A bi a y uni s
)
Sham CIA Sham CIA
SO EVOO
Fig. 7 E ec o die a y EVOO on JNK and p38 MAPKs phospho-
yla ion in hind paws homogena e. The exp ession o phospho yla ed
p o eins was exp essed ela ed o he exp ession o he co esponding
o al p o ein. Da a ep esen mean ± SEM, n = 4. **p < 0.01 and
***p < 0.001 s Sham (SO and EVOO); +p < 0.05, ++p < 0.01 s
SO-CIA
Sham CIA Sham CIA
0.0
0.2
0.4
0.6
0.8
1.0 IκB- αp65
***
++
*
+
SO EVOO
Densi ome y Analysis
(A bi a y uni s
)
Sham CIA Sham CIA
SO EVOO
Fig. 8 Exp ession o IκB-α in cy oplasmic ac ion and p65 NF-κB
in nuclea ac ion o homogena es om hind paws. The exp es-
sion was quan i ied by densi ome y and no malized wi h espec
o β-ac in. Da a ep esen mean ± SEM, n = 4. *p < 0.05 and
***p < 0.001 s Sham (SO and EVOO); +p < 0.05 and ++p < 0.01
s SO-CIA
An i-in lamma o y and join p o ec i e e ec s o ex a- i gin oli e-oil polyphenol
ex ac in expe imen al a h i is☆
Ma ía Ángeles Rosillo
a
, Ma ía José Alca az
b
, Ma ina Sánchez-Hidalgo
a
, José G. Fe nández-Bolaños
c
,
Ca alina Ala cón-de-la-Las a
a
, Ma ía Luisa Fe ándiz
b,
⁎
a
Depa men o Pha macology, Facul y o Pha macy, Uni e si y o Se ille, 41012 Se ille, Spain
b
Depa men o Pha macology and IDM, Uni e si y o Valencia, 46100 Bu jaso , Valencia, Spain
c
Depa men o O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se ille, 41012 Se ille, Spain
Recei ed 11 Ma ch 2014; ecei ed in e ised o m 30 June 2014; accep ed 11 July 2014
Abs ac
The consump ion o ex a i gin oli e oil (EVOO) in Medi e anean coun ies has shown bene icial e ec s. A wide ange o e idence indica es ha phenolic
compounds p esen in EVOO a e endowed wi h an i-in lamma o y p ope ies. In his wo k, we e alua ed he e ec s o EVOO-polyphenol ex ac (PE) in a model o
heuma oid a h i is, he collagen-induced a h i is model in mice. On day 0, DBA-1/J mice we e immunized wi h bo ine ype II collagen. On day 21, mice ecei eda
boos e injec ion.PE(100and200mg/kg)waso allyadminis e edonceaday omdays29 o41 oa h i icmice.Weha edemons a ed ha PEdec easesjoin edema,
cell mig a ion, ca ilage deg ada ion and bone e osion. PE signi ican ly educed he le els o p oin lamma o y cy okines and p os aglandin E
2
in he join as well as he
exp ession o cyclooxygenase-2 and mic osomal p os aglandin E syn hase-1. Ou da a indica e ha PE inhibi s c-Jun N- e minal kinase, p38 and signal ansduce and
ac i a o o ansc ip ion-3. In addi ion, PE dec eases nuclea ac o κB ansloca ion leading o he down- egula ion o he a h i ic p ocess. These esul s suppo he
in e es o na u al die componen s in he de elopmen o he apeu ic p oduc s o a h i ic condi ions.
© 2014 Else ie Inc. All igh s ese ed.
Keywo ds: Rheuma oid a h i is; CIA; EVOO; Polyphenols; In lamma o y esponse
1. In oduc ion
Rheuma oid a h i is (RA) is an au oimmune disease cha ac e -
ized by syno ial in lamma ion and pannus leading o ca ilage and
bone damage. The ea lies e en is ac i a ion o he inna e immune by
exogenous and au ologous an igens and hus au o-an ibodies and
la e high le els o cy okines can be de ec ed yea s be o e clinical
symp oms [1]. A a join le el, he e a e syno ial hype plasia and
massi e in il a ion o immune cells, including CD4
+
T cells, B cells,
na u al kille cells, mac ophages, dend i ic cells, neu ophils and mas
cells [2,3]. A wide ange o e idence has demons a ed he
con ibu ion o cy okines o he ch onici y o in lamma ion and issue
damage in RA. The e o e, he impo ance o p oin lamma o y cy okines
has been con i med by he success o cy okine-di ec ed he apies in RA
[4]. P oin lamma o y cy okines such as in e leukin (IL)-1βand umo
nec osis ac o α(TNFα) ac i a e many signal ansduc ion pa hways
in ol ed in in lamma ion. The mi ogen-ac i a ed p o ein kinase
(MAPK) pa hway includes ex acellula signal- egula ed kinases
(ERK1/2 o p42/p44), c-Jun N- e minal kinases (JNK)1/2/3 and p38,
which a e ac i a ed in he syno ium o pa ien s wi h RA [5].Thejanus
kinase-signal ansduce and ac i a o o ansc ip ion (JAK-STAT) is
ano he ele an in lamma o y pa hway ac i a ed in esponse o
cy okines. In pa icula , o e exp ession o STAT-3 has been epo ed
in syno ial memb anes om RA pa ien s [6].
P oin lamma o ycy okinesac i a edi e en ansc ip ion ac o s o
induce he exp ession o in lamma o y and ca abolic media o s [7].
Nuclea ac o κB(NF-κB) and ac i a ing p o ein-1 (AP-1) a e ac i a ed
in RA syno ium and play an impo an ole in me allop o einase
induc ion [8].NF-κB egula es a wide ange o genes ha con ibu e o
in lamma ion, such as IL-1β,TNFα, IL-6, chemokines and mic osomal
p os aglandin E syn hase-1 (mPGES-1), an e icien downs eam
enzyme unc ionally coupled wi h cyclooxygenase-2 (COX-2).
Biological he apies ha e imp o ed he ea men o ch onic
in lamma o y diseases such as RA. Ne e heless, hese d ugs a e
e ec i e in a ac ion o pa ien s only and ha e o he limi a ions
including a high cos , he equi emen o pa en e al adminis a ion
and impo an side e ec s. The e o e, new he apeu ic s a egies a e
unde in es iga ion. Epidemiological s udies abou consump ion o
unc ional oods, pa icula ly ex a i gin oli e oil (EVOO) in
Medi e anean coun ies, ha e showed impo an bene icial e ec s.
A ailable online a www.sciencedi ec .com
ScienceDi ec
Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
☆
Suppo ed by esea ch g an s SAF2010-22048, RETICEF RD12/0043/
0013and AGL 2011-26949 (Minis e iode Economía y Compe i i idad,ISCIII,
FEDER), PROMETEO II 2014-071 (Gene ali a Valenciana) and P-10AGR-
6609 (Jun a de Andalucía).
⁎Co esponding au ho . Depa men o Pha macology, Facul y o Pha macy,
A .Vicen And ésEs elléss/n,46100Bu jaso ,Valencia,Spain.Tel.: +34 963543284.
E-mail add ess: [email p o ec ed] (M.L. Fe ándiz).
h p://dx.doi.o g/10.1016/j.jnu bio.2014.07.006
0955-2863/© 2014 Else ie Inc. All igh s ese ed.

EVOO, ob ained om he ui o he oli e ee solely by mechanical
o o he physical means unde condi ions ha do no lead o oil
al e a ion, has shown an ioxidan , an i-in lamma o y, immunomod-
ula o y, an ip oli e a i e and an i-apop o ic e ec s [9–15]. Al hough
hese heal h-p o ec i e e ec s ha e been adi ionally asc ibed o i s
high monounsa u a ed a y acid con en [15,16] a wide ange o
e idence indica es ha many o he bene icial e ec s o EVOO in ake
a e due o i s mino highly bioac i e componen s. Among hem,
phenolic compounds such as hyd oxy y osol, y osol and oleu opein
ha e shown an i-in lamma o y and an ioxidan e ec s [17]. Oli e
oil polyphenols ha e also been associa ed wi h neu op o ec i e,
an iaging and an ia he ogenic e ec s and he egula ion o impo an
cellula signaling pa hways. On he o he hand, because o hei
abili y o modula e cell dea h, oli e polyphenols ha e been p oposed
as chemop e en i e and he apeu ic an i-cance agen s [18–21].Ou
esea ch g oup has p e iously demons a ed ha a die made wi h EVOO
en iched wi h hyd oxy y osol, could imp o e in lamma o y p ocesses in
a ch onic coli is model [22]. In addi ion, we e alua ed he p o ec i e
e ec o die a y EVOO-polyphenol ex ac (PE) supplemen a ion in he
in lamma o y esponse associa ed o a ch onic coli is model [23].
Collagen-induced a h i is (CIA) in DBA/1 mice is an animal model
o RA widely used o es po en ial he apeu ic agen s [24]. The
pa hogenesis o CIA is dependen on he hos 's esponse o ype II
collagen challenge and he subsequen gene a ion o an ibodies ha
ecognize collagen- ich join issue [25]. Join in lamma ion in his
model is cha ac e ized by signi ican cellula exuda e and in il a e,
syno i is, and a icula deg ada ion. High le els o p oin lamma o y
cy okines a e de ec ed a he onse o he disease, leading o he
p oduc ion o a wide ange o media o s ele an in CIA pa hogenesis
[26,27]. Taken in o accoun he an i-in lamma o y p ope ies o oli e
oil cons i uen s, we hypo hesized ha PE may con ol he de elop-
men o in lamma ion and a icula lesion. In ac , in ape i oneal
adminis a ion o oleu opein aglycone, a majo cons i uen o he lea es
and unp ocessed oli e d upes, has shown bene icial e ec s in CIA [28],
and a ecen s udy has epo ed ha EVOO die in conjunc ion wi h
physical ac i i y is able o p ese e he a icula ca ilage in an
os eoa h i is model [29].
The p esen s udy was designed o e alua e he e ec s o he o al
adminis a ion o PE in he a h i is model o CIA in mice. In addi ion
o mac oscopic and his ological analyses, we ha e de e mined he
e ec s o PE on he p oduc ion o in lamma o y media o s. In o de o
gain a be e insigh in o i s mechanisms o ac ion, signaling pa hways
we e also explo ed.
2. Ma e ials and me hods
2.1. Ex ac ion o PE
PE was ob ained by he me hod desc ibed by Vazquez Ronce o e al. [30] wi h some
modi ica ions [31]. Fi y g ams o EVOO (Oleoes epa, Se ille, Spain) was ex ac ed wi h
me hanol/wa e (80:20, ol/ ol, 125 ml). The mix u e was mixed wi h a o ex a 5000
g o 1 min and sonica ed o 15 min. A e decan a ion, he me hanolic ex ac was
concen a ed in acuum unde a s eam o ni ogen a b35°C un il i eached a sy up
consis ency; inally, i was lyophilized and s o ed a −80°C.
2.2. Cha ac e iza ion o phenolic compounds in PE
PE (70–75 mg) ex ac ed om EVOO (75 g) was dissol ed in CDCl
3
(750 μl)
o in DMSO-d6 (750 μl) and an p ecisely measu ed olume o he solu ion (550 μl)
was placed in a 5 mm NMR ube o he de ec ion and quan i ica ion o phenolic
compounds. The phenolic mix u e o be analyzed in DMSO-d6 was p e iously
dissol ed in a MeOD-D
2
O 1:1 (1 ml) and concen a ed o d yness a educed p essu e
in o de o exchange hyd oxyl p o ons wi h deu e ium nuclei. The NMR sol en s we e
pu chased om Sigma-Ald ich (S . Louis, MO, USA). NMR expe imen s we e conduc ed
on a B uke A ance III 700 spec ome e , ope a ing a 700.25 M Hz. The p obe
empe a u e was 24.8±1°C. All chemical shi s we e gi en in ppm, and he J couplings
in Hz. The sol en i sel was used as a chemical shi e e ence (7.26 ppm o CHCl
3
, and
2.50 o DMSO-d6).
1
H NMR spec a we e acqui ed wi h he ollowing acquisi ion
pa ame e s: acquisi ion ime 2.3s, elaxa ion delay 5s, spec al wid h o 0–20 ppm,
da a poin s 32k, numbe o scans 32, and line b oadening o 0.3 Hz. Pos acquisi ion
p ocessing included ze o- illing o 64k. The spec a we e phase co ec ed au oma ically
using TOPSPIN and in eg a ion was pe o med manually.
We ha e de e mined he le els o oleocan hal and oleacein ollowing a
modi ica ion o he me hod de eloped by Ka koula e al. [32] o di ec measu emen
o bo h dialdehydes in oli e oil by quan i a i e high esolu ion
1
H NMR in CDCl
3
. The
le els o o he phenolic compounds ha e been de e mined by he p ocedu e desc ibed
by Ch is opho idou and Dais [33].
2.3. Induc ion o CIA
A h i is was induced in male DBA-1/J mice (Jan ie , Le Genes S Isle, F ance) o 11
weeks o age. All s udies we e pe o med in acco dance wi h Eu opean Union
egula ions o he handling and use o labo a o y animals. The p o ocols we e
app o ed by he ins i u ional Animal Ca e and Use Commi ee (Uni e si y o Valencia,
Spain). Bo ine ype II collagen (CII; 2 mg/ml in dilu e ace ic acid; MD Biosciences,
Zü ich, Swi ze land) was emulsi ied in equal olumes o F eund's comple e adju an
(2 mg/ml Mycobac e ium ube culosis, s ain H37Ra; Di co, De oi , MI, USA). On day 0,
DBA-1/J mice we e immunized a he base o he ail wi h 100 µg o bo ine CII. On day
21, mice ecei ed an in ape i oneal boos e injec ion o 100 µg o CII dissol ed in
phospha e-bu e ed saline. Mice we e conside ed o ha e a h i is when signi ican
changes in edness and/o swelling we e no ed in he digi s o in o he pa s o he
paws. Join in lamma ion was sco ed isually in each paw, using a scale o 0–2 whe e
0=unin lamed, 1=mild, 1.5=ma ked and 2=se e e. Sco ing was pe o med by wo
independen obse e s wi hou knowledge o he expe imen al g oups. In addi ion,
pho og aphs and X- ay (Ca es eam MS FX, Gaines ille, FL, USA) images o hind paws
we e ob ained a he end o he expe imen (day 42).
2.4. T ea men g oups
On day 29, animals we e andomized in o con ol and ea men g oups. The s udy
was pe o med in ou g oups o mice (n=10): nai e g oup (NA), CIA g oup (CIA) and
wo ea men g oups: CIA mice wi h PE ea men (PE 100 and 200 mg/kg, o ally, once
a day om days 29 o 41). On day 42 a e immuniza ion mice we e anes he ized, blood
samples we e aken by in aca diac punc u e and animals we e killed by ce ical
disloca ion. Paws we e ampu a ed and p ocessed o ei he his ological analysis o
homogeneiza ion and assessmen o in lamma o y media o s and mechanisms.
2.5. His ological and immunohis ochemical analyses
Knee join s we e ixed in 4% o malin. A e decalci ica ion in 10% EDTA, specimens
we e p ocessed o pa a in embedding. Tissue sec ions (7 μm) we e s ained wi h
hema oxylin and eosin o pe o m his ological analyses. Fo immunohis ochemis y,
he endogenous pe oxidase ac i i y was inhibi ed wi h hyd ogen pe oxide and hen
he sec ions we e incuba ed in no mal ho se se um (Vec as ain Ki ; Vec o
Labo a o ies, Bu lingame, CA, USA) o 20 min o educe nonspeci ic s aining and
successi ely incuba ed wi h abbi an i-COX-2 (Cayman Chemical, Ann A bo , MI, USA)
a dilu ion 1:250 o e nigh a 4°C. La e on, slides we e ea ed wi h an i abbi IgG
an ibody (Vec as ain Ki , Vec o Labo a o ies, Bu lingame, CA, USA) o 30 min and
incuba ed wi h he s ep a idin–pe oxidase complex o he ki o 30 min, a oom
empe a u e. A e incuba ion wi h he pe oxidase subs a e 3,3′-diaminobenzidine
and washing wi h wa e , he sec ions we e coun e s ained wi h hema oxylin. Nega i e
con ol sec ions we e ea ed in he same way bu in he absence o p ima y an ibody.
Posi i e cells and o al cells we e coun ed in i e andom high-powe ields by wo
independen obse e s.
2.6. Enzyme-linked immunoso ben assay
Hind paws (knees) we e homogenized in liquid N
2
wi h 1 ml o A bu e pH 7.46
(10 mM HEPES, pH 8, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM di hio h ei ol (DTT),
5 mM NaF, 1 mM Na
3
VO
4
,1μg/ml leupep in, 0.1 μg/ml ap o inine, and 0.5 mM
phenylme hyl sul onyl luo ide (PMSF)). The issue homogena es we e sonica ed (10 s
h ee imes a 20% wi h a 10-s incuba ion on ice be ween bu s s) in an ul asonic
p ocesso (VC130PB, Sonics & Ma e ials Inc., New own, CT, USA) and cen i uged a
600×g, 15 min a 4°C. Supe na an s we e emo ed and used o de e mina ion o
in lamma o y media o s by enzyme-linked immunoso ben assay (ELISA): PGE
2
(Cayman Chemical, Michigan, USA), TNFαand IL-1β(R&D Sys ems Inc., Minneapolis,
MN, USA), and IL-6 (Diaclone, Besançon Cedex, F ance).
2.7. Wes e n blo
Hind paws we e homogenized in liquid N
2
wi h 1 ml o ice-cold phospha e
bu e (0.01 mM K
2
HPO
4
,KH
2
PO
4
and 0.15 M NaCl) and cen i uged (800×g,10
min a oom empe a u e). Pelle s we e esuspended in hypo onic bu e [1.5
mM MgCl
2
,10mMKCl,0.2mMPMSF,1μg/ml leupep in, 20 mM NaF, 0.5 mM DTT,
1 mM EDTA and 10 mM HEPES, pH 7.9], incuba ed o 10 min on ice and
cen i uged (14,000×g, 30 s a oom empe a u e). Supe na an s (cy oplasmic
p o eins) and pelle s (nuclea p o eins) we e collec ed. Pelle s we e esuspend-
ed in ice-cold low-sal bu e and nuclea p o eins we e eleased by adding a
1276 M.Á. Rosillo e al. / Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
high-sal bu e (20% ol/ olglyce ol,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 leupep in and 20 mM HEPES, pH
7.9). Samples we e incuba ed on ice o 30 min. Soluble nuclea p o eins we e eco e ed
by cen i uga ion (14,000×g, 10 min, 4°C) and p o eins we e s o ed a −80°C. P o ein
concen a ion was de e mined by he B ad o d's colo ime ic me hod [34]. Nuclea
ac ions we e used o s udy p65 p o ein exp ession and cy oplasmic ac ions we e used
o he es o de e mina ions. Aliquo s con ainingequal amoun o p o ein (50 mg) we e
sepa a ed by sodium dodecyl sul a e-polyac ylamide gel elec opho esis (10% gel). The
p o eins we e hen elec opho e ically ans e ed on o a ni ocellulose memb ane and
incuba ed wi h speci ic p ima y an ibodies: abbi polyclonal an i-mPGES-1 (1:200;
Cayman Chemical, USA) abbi polyclonal an i-IκB-α(1:1000; Cell Signalling Technology,
USA), abbi polyclonal p65 (1:200, San aC uzBio echnology, Inc.), abbi polyclonalan i-
p-JNK and abbi polyclonal an i-JNK (1:1000; San a C uz Bio echnology, Inc.), mouse
polyclonal an i-p-p38 and abbi polyclonal an i-p38 (1:1000; San a C uz Bio echnology,
Inc.) and mouse polyclonal an i-p-STAT3 (1:200; San a C uz Bio echnology, Inc.),
o e nigh a 4°C. Memb anes we e washed h ee imes o 15 min and incuba ed wi h
an i- abbi (Pie ce Bio echnology, IL, USA) o an i-mouse (Dako, Ca pin e ia, CA, USA)
ho se adish pe oxidase-labeled seconda y an ibody, o 1–2 h a oom empe a u e. To
p o e equal loading, he blo s we e analyzed o β-ac in exp ession using an an i-β-ac in
an ibody (Sigma–Ald ich). Immunode ec ion was pe o med using enhanced chemilu-
minescence ligh -de ec ing ki (Supe Signal Wes Fem o Chemiluminescen Subs a e,
Pie ce, IL, USA). Densi ome ic da a we e s udied ollowing no maliza ion o he con ol
(housekeeping gene). The signals we e analyzed and quan i ied by ImageJ so wa e
(Be hesda, MD, USA).
2.8. Da a analysis
All alues in he igu es and ex a e exp essed as a i hme ic means±s anda d
e o (S.E.M.). Da a we e e alua ed wi h G aph Pad P ism Ve sion 5.04 so wa e. The
s a is ical signi icance was e alua ed by wo-way ( ime cou se) o one-way ( es o
pa ame e s) analysis o a iance (ANOVA), ollowed by Bon e oni's es . P alues b.05
we e conside ed s a is ically signi ican .
3. Resul s
3.1. Chemical composi ion o PE
Table 1 shows he esul o quali a i e and quan i a i e analyses o
PE. Nine di e en phenolic compounds ha e been iden i ied. Oleocan hal
and oleacein a e he main componen s o PE.
3.2. E ec o PE on es ablished CIA
The ime-cou se o a h i ic sco e (Fig. 1A) indica es ha con ol
CIA mice showed a p og essi e de elopmen o clinical symp oms.
The se e i y o a h i is in he g oups ea ed wi h PE (100 and
200 mg/kg) was lowe han ha o he a h i ic con ol g oup wi h
signi ican esul s o he highes dose on days 39 and 41. Fig. 1B
shows ep esen a i e pho og aphs o hind paws om he di e en
g oups. In addi ion, X- ay images (Fig. 1C) e ealed he p esence o
join edema and ocal bone e osions in hind paws o CIA mice,
whe eas in animals ea ed wi h PE, he in lamma ion was lowe .
In e es ingly, in he PE200 g oup, he in eg i y o bone was simila o
ha o naï e mice. Con ol CIA mice exhibi ed his ological changes
indica i e o se e e a h i is, wi h ex ensi e in il a ion o in lamma-
o y cells in o a icula issues, exuda ion in o he syno ial space,
syno ial hype plasia and ca ilage e osion (Fig. 2). These his ological
ea u es we e less appa en in mice ea ed wi h PE (200 mg/kg).
3.3. E ec o PE on join in lamma o y media o s
We also explo ed whe he PE could modi y he p oduc ion o
p oin lamma o y cy okines and PGE
2
. A he end o he expe imen al
pe iod (day 42), he le els o IL-1β, TNFαand IL-6 we e de e mined in
hind paw homogena es. As shown in Fig. 3, animals ea ed wi h PE
(100 o 200 mg/kg) showed a signi ican educ ion in join le els o all
p oin lamma o y cy okines. In addi ion, a signi ican inc ease in PGE
2
le els in he paws o a h i ic con ol animals wi h espec o naï e
mice was ound (Fig. 3). T ea men wi h PE educed he le els o his
eicosanoid, which eached s a is ical signi icance a bo h doses.
3.4. E ec o PE on COX-2 and mPGES-1 exp ession
The e ec o PE on COX-2 p o ein exp ession was de e mined by
immunohis ochemis y o knee join sec ions (Fig. 4A). We obse ed
a signi ican inc ease in he pe cen age o COX-2-posi i e cells in
ca ilage o a h i ic con ol g oup (CIA), wi h espec o naï e
animals. In he g oup ea ed wi h PE (200 mg/kg), he immuno e-
ac i i y o his p o ein was signi ican ly educed. COX-2 exp ession
was accompanied by a signi ican inc ease in mPGES-1 p o ein
exp ession, which was de e mined by Wes e n blo , in he paws o
CIA con ol mice wi h espec o naï e animals (Fig. 4B). T ea men
wi h PE signi ican ly dec eased he exp ession o his p o ein.
3.5. E ec o PE on p38 and JNK phospho yla ion
p38 and JNK play an impo an ole in in lamma ion and issue
damage in RA [5,35]. We in es iga ed he e ec o PE on he ac i a ion
o JNK and p38 in he CIA model by wes e n blo using phosphospeci ic
an ibodies. The de elopmen o CIA induced s ong MAPK phospho yla ion
in join s (Fig. 5). Howe e , PE a bo h doses signi ican ly dec eased he
phospho yla ion o JNK and p38.
3.6. E ec o PE on p-STAT3 p o ein exp ession in paw homogena e
p-STAT3 p o ein exp ession was e alua ed by Wes e n blo
analysis o hind paw homogena es. Fig. 6 shows ha a h i is s ongly
induced STAT3 phospho yla ion, as obse ed in CIA con ol mice wi h
espec o naï e animals. T ea men wi h PE a 200 mg/kg and, o a
lowe ex en , a 100 mg/kg, signi ican ly educed he exp ession o p-
STAT3 in a h i ic mice.
3.7. E ec s o PE on NF-κB signaling pa hway
We in es iga ed he e ec o PE on IκB-αexp ession in
cy oplasmic ex ac s o mice join s. As shown in Fig. 7, he exp ession
o IκB-αwas signi ican ly educed in a h i ic con ol mice (CIA)
compa ed wi h naï e mice. The e ec o a h i is on IκB-αwas
blocked by PE ea men a he dose o 200 mg/kg. The educ ion in
IκB-αexp ession obse ed in CIA mice is consis en wi h an inc ease
in IκB-αdeg ada ion hus allowing he ansloca ion o NF-κB in o he
nucleus o bind speci ic DNA sequences leading o he ansc ip ion o
p oin lamma o y genes. The e o e, CIA mice showed an inc eased
exp ession o p65 NF-κB in nuclea ex ac s o join s which was
signi ican ly inhibi ed by PE a 200 mg/kg.
4. Discussion
In his s udy, we ha e demons a ed ha o al adminis a ion o PE
is able o down- egula e he a h i ic p ocess in he CIA model o RA.
PE showed an i-in lamma o y e ec s wi h educ ions in join edema
and mig a ion o in lamma o y cells. In addi ion, PE p o ec ed join s
agains ca ilage al e a ions and bone e osion. Acco ding o p e ious
Table 1
Chemical cons i uen s o PE
Phenolic compound μmol/100 g ppm
F ee hyd oxy y osol 2.25 3.47
F ee y osol 5.21 7.20
Oleacein 9.62 30.82
Oleocan hal 10.43 31.74
Oleu opein aglycone monoaldehyde (5R,8S,9R) 1.49 5.63
Ligs oside aglycone monoaldehyde (5R,8S,9R) 3.88 14.06
Apigenin 0.56 1.52
Lu eolin 1.26 3.61
Ace oxypino esinol 1.92 8.00
1277M.Á. Rosillo e al. / Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
epo son hebiologicalp ope ies o oli eoilcons i uen s[17,18,28,9],
he an i-in lamma o y e ec s o PE a e likely dependen on he mix u e
o di e en phenolic compounds.
The p og ession o a h i is is associa ed wi h sus ained p oduc-
ion o p oin lamma o y cy okines [4]. We ha e shown in he CIA
model ha PE con ols he local le els o hese cy okines d i ing he
p oduc ion o di e en in lamma o y and ca abolic media o s.
P oin lamma o y cy okines induce u he cy okines, chemokines,
eicosanoids and eac i e oxygen species (ROS) ampli ying he
in lamma o y esponse [36]. The e o e, COX-2 and mPGES-1, en-
zymes esponsible o he o e p oduc ion o PGE
2
in in lamma ion,
a e up- egula ed [37] con ibu ing o he p og ession o RA h ough
EP
4
ecep o ac i a ion [38]. We ha e shown ha PE educes PGE
2
le els which would be dependen on he down- egula ion o COX-2
and mPGES-1 exp ession in he join . Cy okines also induce he
exp ession o ma ix me allop o einases a he pannus ca ilage
junc ion leading o ca ilage deg ada ion [39,40]. In addi ion, TNFα
s imula es os eoclas ogenesis [41], supp esses he ec ui men o
os eoblas s and inhibi s he exp ession o ma ix genes [42], whe eas
IL-6 inc eases os eoclas numbe s in abecula bone [43]. The e o e,
inhibi ion o p oin lamma o y cy okine p oduc ionby PE would esul
in he educ ion o he in lamma o y esponse and issue damage.
MAPKs egula e he syn hesis o chemokines, cy okines, adhesion
molecules and PGs in ol ed in in lamma ion. In addi ion, hey
media e he induc ion o ma ix me allop o einases esponsible o
ca ilage b eakdown. JNK1/2 and p38 a e also in ol ed in os eoclas
di e en ia ion and hus he ac i a ion o hese enzymes may
con ibu e o bone des uc ion [35]. In pa icula , p38 plays a key
0
1
2
3
4
29 31 34 36 39 41
A
Days
Naï e
CIA
PE100
PE200
Clinical sco e
**
*
++
++ ++
Naï e CIA
PE100 PE200
BC
Naï e CIA
PE100 PE200
Fig. 1. Time cou se o he a h i is mac oscopic sco e (A), ep esen a i e pho og aphs (B) and X- ay images (C) o hind paws a he end o he expe imen (day 42). Naï e, non-a h i ic
mice;CIA,con ola h i icg oup;PE 100,a h i icmice ea edwi hPE(100mg/kg/dayp.o. omdays29 o41 a e immuniza ion);PE 200,a h i ic mice ea edwi hPE(200mg/kg/day
p.o. om days 29 o 41 a e immuniza ion). Da a ep esen mean±S.E.M. n=10. +Pb.05, ++Pb.01 s. naï e; *Pb.05, **Pb.01 s. CIA. A ows indica e ocal bone e osion.
Naï e CIA PE 200
Fig. 2. His ological analysis o he on al sec ions o knee join s on day 42. Sec ions we e s ained wi h hema oxylin and eosin. O iginal magni ica ion ×100. Naï e, nona h i ic mice;
CIA, con ol a h i ic g oup; PE 200, a h i ic mice ea ed wi h PE (200 mg/kg/day p.o. om days 29 o 41 a e immuniza ion). The images a e ep esen a i e o a leas six
expe imen s pe o med on di e en days.
1278 M.Á. Rosillo e al. / Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
ole in RA [44]. JAKs bind o in acellula domains o IL-6 o in e e on
ecep o s and ca alyze ligand-induced phospho yla ion o hemsel es
and o in acellula y osine esidues. Phospho yla ion o STATs
on y osine esidues esul s in he o ma ion o STAT dime s ha
ansloca e in o he nucleus o bind speci ic DNA sequences.
This pa hway can be egula ed by di e en p o ein kinases such as
MAPK ha phospho yla e STATs on se ine esidues o po en ia e
STAT-ac i a ing s imuli [45]. STAT3 may con ibu e o he ch onici y
o in lamma ion in expe imen al a h i is [46]. Ou da a indica e ha
PE adminis a ion is able o educe he phospho yla ion o JNK,
p38 and STAT3, sugges ing ha PE could con ol he ac i a ion o
hese impo an signaling pa hways du ing he a h i ic p ocess.
The NF-κB pa hway is in ol ed in he ansc ip ion o many
in lamma o y genes [47]. Cellula s imula ion by p oin lamma o y
cy okines induces he ec ui men o cos imula o y molecules, such
as TNF ecep o -associa ed ac o (TRAF) leading o he ac i a ion o
NF-κB-inducing kinase. This p o ein induces IκB kinase ac i a ion
esul ing in he phospho yla ion o IκB[6], which is ollowed by
ubiqui ina ion and p o eoly ic deg ada ion hus allowing he elease
o NF-κB o en e he nucleus and egula e gene ansc ip ion [48].
0
1000
2000
3000
4000
5000
0
20
40
60
80
0
1000
2000
3000
4000
++
** **
IL-1β
pg/g issue
++
** **
pg/g issue
TNFα
++
***
pg/g issue
IL-6
Naï e CIA PE 100 PE 200 Naï e CIA PE 100 PE 200
Naï e CIA PE 100 PE 200
0
10
20
30
40
Naï e CIA PE 100 PE 200
PGE2
ng/g issue
++
**
*
Fig. 3. Le els o TNFα, IL-1β, IL-6 and PGE
2
in paw homogena es. These in lamma o y media o s we e de e mined by ELISA. Da a ep esen mean±S.E.M., n=6. ++Pb.01 s. naï e;
*Pb.05, **Pb.01 s. CIA.
BA
0
20
40
60
80
100 ++
**
Naï e CIA PE 200
% Posi i e cells
COX-2
0.0
0.2
0.4
0.6
0.8
1.0
Naï e CIA PE 200
Naï e CIA PE 100 PE 200
Naï e CIA PE 100 PE 200
++
** **
mPGES-1
mPGES-1/β-ac in
Fig. 4. Exp ession o COX-2 (A) and mPGES-1 (B) in hind paws. COX-2 was de e mined by immunohis ochemis y and exp essed as pe cen age o posi i e cells. mPGES-1 was
de e mined by Wes e n blo , quan i ied by densi ome y and no malized wi h espec o β-ac in. Da a ep esen mean±S.E.M., n=4. ++Pb.01 s. naï e; **Pb.01 s. CIA.
1279M.Á. Rosillo e al. / Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
Ou esul s indica e ha PE enhances he le els o he inhibi o y
p o ein IκB-αleading o he educ ion o NF-κB nuclea ansloca ion.
This mechanism may be esponsible o he down- egula ion o
p oin lamma o y cy okines in he join du ing he CIA p ocess in he
animals ea ed wi h PE. P oin lamma o y cy okines and ROS could ac
as media o s o NF-κB ac i a ion [49]. As demons a ed in p e ious
s udies, PE inhibi s he gene a ion o ROS [50] besides i s e ec on
p oin lamma o y cy okines, sugges ing ha bo h mechanisms could be
in ol ed in he con ol o NF-κB ansloca ion.
In conclusion, o s udy has demons a ed he an i-in lamma o y
and join p o ec i e e ec s o PE om EVOO, which would be ela ed
o he inhibi ion o ele an signaling pa hways such as NF-κB, STAT-
3, JNK and p38 con olling he p oduc ion o in lamma o y media o s.
Ou da a also show he in e es o na u al die componen s in he
de elopmen o he apeu ic p oduc s o a h i ic condi ions.
Acknowledgmen
M.A.R. and M.L.F. pe o med he expe imen s and da a analysis.
M.S.H. and J.G.F.B. pe o med pa o he analysis. C.A. and M.J.A.
designed he s udy and p epa ed he manusc ip . All au ho s ead and
app o ed he inal con en o he manusc ip . The au ho s decla e ha
hey ha e no con lic s o in e es .The au ho s g a e ully acknowledge
he assis ance o Cen e o Technology and Inno a ion Resea ch,
Uni e si y o Se ille (CITIUS) and he I+D+i Oleoes epa SAC
Depa men who ga e us he EVOO.
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0.0
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0.0
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**
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by densi ome y and no malized wi h espec o β-ac in. Da a ep esen mean±S.E.M.,
n=4. ++Pb.01 s. naï e; *Pb.05, **Pb.01 s. CIA.
0.0
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1.5
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++ ++
**
**
IκB-α
p65
Naï e CIA PE 100 PE 200
P o ein/β-ac in
Fig. 7. Exp ession o IκBαin cy oplasmic ac ion and p65 NF-κB in nuclea ac ion o
homogena es om hind paws. The exp ession was quan i ied by densi ome y and
no malized wi h espec o β-ac in. Da a ep esen mean±S.E.M., n=4. ++Pb.01 s.
naï e; **Pb.01 s. CIA.
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1281M.Á. Rosillo e al. / Jou nal o Nu i ional Biochemis y 25 (2014) 1275–1281
CAPÍTULO III
P e en i e e ec s o die a y
hyd oxy y osol ace a e, an ex a
i gin oli e oil polyphenol in mu ine
collagen-induced a h i is.
(Rosillo e al. Mol Nu Food Res. 2015. In p ess)
Capí ulo 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
, Ma ina Sánchez-Hidalgo
1
, Alejand o González-Benjumea
2
, José G.
Fe nández-Bolaños
2
, E ik Lubbe s
3
, Ca alina Ala cón-de-la-Las a
1
1
Depa amen o de Fa macología, Facul ad de Fa macia, Uni e sidad de Se illa, España
2
Depa amen o de Química O gánica, Facul ad de Química, Uni e sidad de Se illa, España
3
Depa amen o de Reuma ología, E asmus MC, Uni e sidad E asmus, Ro e dam, Holanda
RESUMEN
An eceden es del ema: En la AR, a ni el de las a iculaciones se p oduce hipe plasia sino ial y
una in il ación masi a de células inmunes, incluyendo células T, células B, mac ó agos,
neu ó ilos, células dend í icas, en e o as. Aunque la pa ogenia de la AR sigue sin conoce se
en su o alidad, a ances en la in es igación de la en e medad han demos ado que el aumen o
de los ni eles de ci ocinas p oin lama o ias como TNF-α, IL-1β, IL-6 e IFN-γ y el aumen o de los
ni eles de me alop o easas (MMP) en el sino io de las a iculaciones de los pacien es a í icos,
juegan un papel undamen al en la e osión del ca ílago a icula y ósea. En la exp esión génica
de es os mediado es p oin lama o ios, pa icipan di e sas ías de señalización celula en e las
que cabe des aca las MAPKs, JAK/STAT o NF-κB, las cuales se encuen an ac i adas en el sino io
de pacien es con AR. La ac i ación de es as ías de señalización, ambién es án implicadas en la
exp esión de genes de p o eínas p oin lama o ias, como COX-2 o m-PGES-1, ambas elacionadas
con el aumen o de la exp esión del ecosanoide p oin lama o io PGE
2.
Po o o lado, el ac o
nuclea N 2, es una pieza cla e en la ansc ipción de dis in as enzimas an ioxidan es como la
hemo oxigenasa 1 (HO-1), se ha demos ado que la ac i ación de la exp esión génica de es a
p o eína es capaz de modula la espues a in lama o ia. A pesa de los a ances en la e apia
a macológica pa a el a amien o de la AR, supone cie as limi aciones, como sus e ec os
ad e sos, es po lo que en los úl imos años ha c ecido el in e és en la u ilización de suplemen os
die é icos y nu acéu icos, ca en es de los e ec os ad e sos que acompaña a la a maco e apia
clásica. Es udios epidemiológicos y expe imen ales han pues o de mani ies o el papel
bene icioso que eje cen dis in os poli enoles de la die a en la AR. AOVE, es ico en una a iedad
de compues os enólicos, en e los que se encuen a el ace a o de hid oxi i osol (Ac-HT), que ha
demos ado ene e ec os an ioxidan es y an iin lama o ios.
Capí ulo III
82
Obje i os: En base a los an e io es an eceden es, nos plan eamos e alua la in luencia de una
die a en iquecida con Ac-HT, en un modelo de a i is expe imen al inducida po colágeno ipo
II en a ones.
Ma e ial y Mé odos: Ra ones DBA-1/J ecién des e ados, ue on dis ibuidos en cua o g upos
expe imen ales: (1) Ra ones sanos alimen ados con die a es ánda (CS), (2) Ra ones a í icos
alimen ados con di a es ánda (CIA), (3) Ra ones a í icos alimen ados con die a en iquecida
con hid oxi i osol (HT) (CIA-HT), (4) Ra ones a í icos alimen ados con die a en iquecida con Ac-
HT (CIA- Ac-HT). Después de seis semanas, la a i is ue inducida. El día 0, los a ones ue on
inmunizados con una inyección en la base de la cola de 100 mg de colágeno II. El día 21,
ecibie on una inyección de e ue zo in ape i oneal, con la misma can idad de colágeno. El
g ado de desa ollo de la en e medad ue alo ado de o ma isual siguiendo una escala de 0-2,
dónde 0=sin in lamación; 1=in lamación le e; 1.5=in lamación ma cada; 2=in lamación se e a y
según el es udio his ológico de las a iculaciones. Los ni eles en sue o de IgG1, IgG2a, COMP y
MMP-3 así como los ni eles de ci ocinas p oin lama o ias como TNF-α, IL-1β, IFN-γ, IL-6 e IL-17
en el homogenado de pa a ue on de e minados median e la écnica de ELISA. Los cambios en
la exp esión p o eica de HO-1, N 2 y de las p o eínas implicadas en las ías de señalización
celula MAPKs, JAK/STAT y NF-κB ue on es udiadas median e wes e n blo .
Resul ados: la die a en iquecida con Ac-HT, edujo de o ma signi ica i a el edema a icula y la
des ucción del ca ílago, p e iniendo el desa ollo de la AR. Los ni eles sé icos de las IgG1 y 2 a
an o mu inas como bobinas, así como los ni eles de COMP y MMP-3, los encon amos
disminuidos en aquellos animales alimen ados con la die a en iquecida con Ac-HT. La exp esión
de las ci ocinas p oin lama o ias TNF-α, IL-1β, IFN-γ, IL-6 e IL-17 ue meno el ejido a icula de
los a ones a í icos que ecibie on la die a en iquecida con el ace a o. El Ac-HT p odujo una
inhibición en la os o ilación de la p o eína STAT-3, así como de las p o eínas MAPKs e inhibió la
anslocación al núcleo de la p o eína p 65 NF-κB, p e iniendo la deg adación de la p o eína
inhibi o ia IκB-α. Además, Ac-HT ue capaz de aumen a la exp esión p o eica de N 2 y HO-1.
Conclusión: Es os esul ados ponen de mani ies o, po p ime a ez, el e ec o an iin lama o io
de una die a en iquecida con Ac-HT en un modelo de a i is expe imen al en a ones, que
es u o acompañado po una educción de los ma cado es COMP y MMP-3 en sue o. Los
mecanismos implicados pod ían es a elacionados con una egulación de la espues a de las
células B, una ac i ación de la ía N 2/HO-1 y la inhibición de la ac i ación de dis in as ías de
señalización, como son JAK/STAT, MAPKs y NF- κB, implicadas en la p oducción de mediado es
in lama o ios como ci ocinas y PGE
2.
Concluyendo que un suplemen o de AC-HT en la die a,
6M. A. Rosillo e al. Mol. Nu . Food Res. 2015, 0,1–10
Figu e 4. (A) Exp ession o
COX-2 and mPGES-1 in hind
paws. COX-2 and mPGES-1
we e de e mined by wes e n
blo , quan i ied by densi ome y
and no malized wi h espec
o ␤-ac in. (B) Measu emen
o PGE2 le els in hind paw
homogena es. These we e
de e mined by ELISA ki s. Da a
ep esen mean ±SEM, n=
4. **p<0.01 and ***p<0.001
e sus na¨
ı e; ++p<0.01 and
+++p<0.001 e sus CIA;
&&p<0.01 e sus CIA-HTy.
HTy die showed i s ine ec i eness along he expe imen al
pe iod.
3.9 E ec s o die a y HTy-Ac on NF-␬B signaling
pa hway
We also in es iga ed he e ec s o HTy-Ac on IkB-␣deg a-
da ion in cy oplasmic ex ac s om mice join s. As shown
in Fig. 6B, I␬B-␣exp ession was signi ican ly educed in
CIA-induced a h i is mice when compa ed wi h na¨
ı e mice
(p<0.001 e sus na¨
ı e). The I␬B-␣exp ession obse ed in
CIA mice was consis en wi h an inc ease in I␬B-␣deg a-
da ion hus allowing NF-␬B ansloca ion in o he nucleus
o bind speci ic DNA sequences leading o p o-in lamma o y
genes ansc ip ion. Acco ding o he esul s ob ained, he
e ec obse ed in a h i ic con ol g oup on I␬B-␣p o ein
deg ada ion was p e en ed by die a y HTy-Ac ea men . On
he con a y, he nuclea p65 p o ein le els we e signi i-
can ly inc eased in CIA g oup (p<0.001 e sus na¨
ı e) whe eas
Figu e 5. (A) Changes in p-STAT3 p o ein exp ession in hind paws homogena e. The exp ession o p-STAT3 was quan i ied by densi ome y
and no malized wi h espec o ␤-ac in. (B) HTy-Ac die a y up egula ed HO-1 and N 2 p o ein exp ession in hind paws homogena e. The
exp ession was quan i ied by densi ome y and no malized wi h espec o ␤-ac in. Da a ep esen mean ±SEM, n=4. ***p<0.001 e sus
na¨
ı e; +p<0.05 and ++p<0.01 e sus CIA; &p<0.05 and &&p<0.01 e sus CIA-HTy.
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Mol. Nu . Food Res. 2015, 0,1–10 7
Figu e 6. (A) E ec o die a y HTy-Ac on JNK, p38 and ERK (1/2) MAPKs phospho yla ion in hind paws homogena e. The exp ession o
phospho yla ed p o eins was exp essed ela ed o he exp ession o he co esponding o al p o ein. (B) Exp ession o I␬B-␣in cy oplasmic
ac ion and p65 NF-␬B in nuclea ac ion o homogena es om hind paws. The exp ession was quan i ied by densi ome y and no malized
wi h espec o ␤-ac in. Da a ep esen mean ±SEM, n=4. *p<0.05 and ***p<0.001 e sus na¨
ı e; +p<0.05, ++p<0.01 e sus CIA; &p<0.05
and &&p<0.01 e sus CIA-HTy.
die a y HTy-Ac ea men p e en ed he CIA-induced nuclea
ansloca ion le el o p65 in paw homogena e in compa ison
wi h hose a h i ic animals ed wi h SD (p<0.01 e sus CIA)
(Fig. 6B) a oiding he NF-kB-media ed ansc ip ional ac i a-
ion. HTy die ailed o modi y I␬B-␣deg ada ion and nuclea
p65 ansloca ion in CIA mice.
4 Discussion
Ou indings, ha e shown, o he i s ime, ha die a y HTy-
Ac en ichmen was able o p e en and down- egula e he
a h i ic p ocess in he CIA model o RA. This model is com-
monly used o in es iga e ele an pa hogenic mechanisms
o RA as well as new an ia h i ic ea men s [19]. HTy-Ac
was desc ibed o he i s ime in oli e oil by B enes e al.
[20] and is ound in mos Spanish i gin oli e oils. Mo eo e ,
ecen ly, i was epo ed by Ma eos e al. [21] ha HTy-Ac is
mo e soluble in he lipophilic phases han HTy, due o he
p esence o he es e g oup, which was demons a ed in a
Caco-2 cell model. Thus, his inc eased lipophilici y sugges s
ha HTy-Ac is be e abso bed ac oss in es inal epi helial
cell monolaye s han ee HTy [22]. CIA induc ion esul ed
in he de elopmen o a p onounced syno i is associa ed
wi h ca ilage deg ada ion and bone e osion [23]. Howe e ,
die a y HTy-Ac supplemen a ion could imp o e he a h i-
is sco e which was co ela ed wi h a mino mig a ion o
in lamma o y cells in o a icula issues in addi ion o a
ma ked educ ion o join edema, syno ial hype plasia and
ca ilage e osion in compa ison wi h hose animals ed wi h
SD and HTy en iched die s.
A ole o humo al immuni y in he pa hogenesis o au-
oimmune a h i is is sugges ed by e idence de i ed om
animal models as well as pa ien s wi h RA. An ibody/an igen
complexes a e abundan ly ound in he join s o RA pa ien s
and a e belie ed o play a ole in igge ing he join in lamma-
ion [24]. CIA pa hogenesis is cha ac e ized by he gene a ion
o an i-CII an i-bodies. All iso ypes (IgG1, IgG2a and IgG2b)
o an i-CII an ibodies we e a h i ogenic, wi h he IgG1 and
IgG2b iso ypes as he domina ing a h i ogenic an ibodies
[25]. In he p esen s udy, die a y HTy-Ac supplemen a ion
signi ican ly dec eased se um le els o IgG1-and IgG2a-an i-
CII an ibodies in CIA mice. These esul s sugges a po en ial
ole o HTy-Ac in egula ing B cell esponses, which may be
in pa , esponsible o i s an i-a h i ic e ec s.
The de elopmen and p og ession o RA is closely ela ed
o an imbalance o cy okine ne wo k. In RA syno ium, ele-
a ed le els o p o-in lamma o y cy okines such as TNF-␣,IL-
1␤, IL-6, IL-17 and INF-␥a e p oduced by mac ophages and
syno ial ib oblas s. These p o-in lamma o y cy okines bo h
di ec ly and indi ec ly exe hei e ec s h ough he p oduc-
ion o addi ional p o-in lamma o y cy okines, chemokines
and MMPs a he pannus ca ilage junc ion leading o ca -
ilage deg ada ion [26–28]. In addi ion, TNF-␣s imula es
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8M. A. Rosillo e al. Mol. Nu . Food Res. 2015, 0,1–10
os eoclas ogenesis [29], supp esses he ec ui men o os-
eoblas s and inhibi he exp ession o ma ix genes [30],
whe eas IL-6 inc eases os eoclas numbe s in abecula bone
[31]. Besides, IL-17 is a T cell-de i ed cy okine able o induce
he elease o IL-8 and IL-6, and plays a conside able ole in
he addi i e/syne gis ic e ec s induced by TNF-␣and IL-1␤
[32].
The p esen s udy showed ha a h i ic mice ed wi h
HTy-Ac en iched die had dec eased IL-1␤, IL-6, IFN-␥, IL-17
and TNF-␣le els in paw homogena e in compa ison wi h
CIA mice ed wi h SD and HTy en iched die . These esul s
indica e ha die a y HTy-Ac exe s an i-in lamma o y ac i i-
ies by he blockage o IL-1␤,IL-6,IFN-␥, IL-17 and TNF-␣
p oduc ion.
COMP, a p ominen non-collagenous componen o ca -
ilage, accoun s o app oxima ely 1% o he we weigh o a -
icula issue and shows g ea po en ial as a biological ma ke
o ca ilage me abolism in a h i is [33]. Inc eased agmen s
o COMP ha e been epo ed in pa ien s wi h os eoa h i is,
RA and join inju y, hus and he moni o ing o COMP le els
in syno ial luid o se um has been sugges ed o be a help ul
me hod o assessing he p esence and p og ession o a h i-
is. In ac , se um COMP is educed in RA pa ien s in emis-
sion [33]. In addi ion, COMP is a pu a i e subs a e o MMPs.
In pa icula , MMP-3, is a p o einase sec e ed by syno ial i-
b oblas s and chond ocy es. I s syn hesis and ac i a ion is
induced by a ious ac o s, including p o-in lamma o y cy-
okines and Toll-like ecep o ligand. I s ac i i y is associa ed
wi h highe join damage in RA [34] and CIA [4] and esul s in
deg ada ion o agg ecan co e p o ein, ca ilage link p o ein,
ib onec in and collagen ypes IV, VII, IX and XI. Likewise,
se um MMP-3 le el is sugges ed as a p edic o o join de-
s uc ion in ea ly RA o es ablished RA and should be used in
associa ion wi h usual in lamma o y ma ke s o ollow he -
apy e iciency [35]. Ou da a a e in ag eemen wi h abo e
s udies and showed ha high se um COMP and MMP-3 le -
els we e associa ed wi h disease ac i i y and join p og ession
in CIA mice, by con as he p oduc ion o bo h ca ilage and
syno ial bioma ke s we e signi ican ly inhibi ed by he HTy-
Ac en iched die in CIA mice.
COX-2 and mPGES-1, enzymes esponsible o he o e -
p oduc ion o PGE2in in lamma ion, a e up- egula ed [36]
con ibu ing o he p og ession o RA h ough EP4 ecep-
o ac i a ion [7]. We ha e shown ha die a y HTy-Ac sup-
plemen a ion educed PGE2 le els which could be possibly
due o dec eased exp ession o bo h COX-2 and mPGES-1
in he join . The e o e, egula ion o hese p o-in lamma o y
bioma ke s by HTy-Ac could ep esen a po en ial molecula
a ge suscep ible o HTy-Ac modula ion, which has no been
demons a ed p e iously.
Signal ansduc ion pa hways closely in ol ed in in lam-
ma ion include he MAPKs, JAK-STAT and NF-␬B pa hways
[37,38]. In ac , NF-␬B nuclea ansc ip ion ac o plays a pi -
o al ole in he de elopmen and ac i a ion o Th-1 esponses
[39] and is esponsible in addi ion o MAPKs o COX-2 up-
egula ion [40]. We in es iga ed whe he he ac i a ion o
NF-␬B signaling pa hway was inhibi ed by HTy-Ac die . Ou
da a a e in ag eemen wi h Sanchez-Fidalgo e al. ha showed
ha HTy-Ac inc eased he inhibi o y p o ein IkB-␣and e-
duced p65 ansloca ion, indica ing ha die a y HTy-Ac in-
hibi ed he NF-␬B ac i a ion by blocking I␬B-␣deg ada ion
in dex an sul a e sodium -induced coli is in mice [16].
MAPK amily membe s, including p38 kinases, ERKs 1
and 2 and JNKs, a e in ol ed in many impo an cell p o-
cesses, mainly he egula ion o he syn hesis o chemokines,
cy okines, adhesion molecules and PGs in ol ed in RA [6,41].
Besides JNK MAPK modula es MMPs p oduc ion by syn-
o ial ib oblas s and d i es os eoclas di e en ia ion in RA
[42]. In pa icula , p38 MAPK egula es MMP-3 induc ion
in ib oblas s [43] and os eoclas di e en ia ion [44]. Mo e-
o e , MAPKs phospho yla e he JAK-STAT impo an in p o-
in lamma o y cy okine-media ed signaling pa hways such as
Th17 cell di e en ia ion, leading o STAT-3 ac i a ion by
phospho yla ion on y osine esidues esul ing in he o -
ma ion o STAT dime s ha ansloca e in o he nucleus o
bind speci ic DNA sequences [45]. In his line, STAT-3 o e -
exp ession has been also de ec ed in syno ial memb anes
om RA pa ien s and i has been epo ed o con ibu e o
he ch onici y o CIA induced a h i is model [4, 46]. Ou
indings a e in conco dance wi h abo e epo s and showed
ha p38 and JNK MAPKs phospho yla ion we e inc eased
in CIA con ol mice. Simila ly, STAT-3 o e exp ession was
also e idenced in RA syno ium o con ol CIA mice and was
posi i ely ela ed o he se e i y o syno i is, whe eas die a y
HTy-Ac supplemen a ion educed signi ican ly bo h MAPKs
and STAT-3 ac i a ion a ansc ip ional le el. Collec i ely,
ou da a sugges ha die a y HTy-Ac may ep ess IL-17 p o-
duc ion in e e ing nega i ely wi h JNK, p-38, p-ERK MAPKs
and STAT-3 signaling pa hways.
N 2, is a edox-sensi i e ansc ip ion ac o and binds
o an ioxidan esponse elemen s (ARE) loca ed in he p o-
mo e egions o many de oxi ying/an ioxidan genes, includ-
ing HO-1 [47]. In in lamma o y condi ions, HO-1 exp ession
p o ein could be pa o an adap i e mechanism o limi cy-
o oxici y ia se e al mechanisms including sca enging o
eac i e oxygen o ni ogen species, egula ion o cell p o-
li e a ion and p e en ion o apop osis. Likewise, i has been
epo ed ha HO-1 de iciency in mice esul s in a ch onic
in lamma o y s a e [9]. Thus, N 2 egula es edox s a us and
plays key oles in cellula de ense by enhancing he emo al
o eac i e oxygen species [48]. Fu he mo e, i has been doc-
umen ed ha de iciency o N 2 accele a es he e ec o phase
o a h i is and agg a a es join disease [49]. In he p esen
s udy, in conco dance wi h Ka a as e al. [50], exp essions o
N 2 and HO-1 we e dec eased in he a h i is g oup, by con-
as die a y HTy-Ac could es o e N 2 and HO-1 exp essions
con e ing a ole o N 2/HO-1 signaling in he bene icial e -
ec s o HTy-Ac in his mu ine model o RA.
Collec i ely, ou s udy has demons a ed o he i s ime
he an i-in lamma o y e ec s o die a y HTy-Ac in he CIA
model, which we e accompanied by an impo an educ ion
o he se um syno ial and ca ilage bioma ke s COMP and
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Mol. Nu . Food Res. 2015, 0,1–10 9
MMP-3. The mechanisms unde lying hese p o ec i e e ec s
could be ela ed o he egula ion o B cell esponses, ac i a-
ion o he N 2/HO-1 signaling and he inhibi ion o ele an
signaling pa hways such as JAK-STAT, MAPKs and NF-␬B
hus con olling he p oduc ion o in lamma o y media o s
such as Th1 cy okines and PGE2. We concluded ha HTy-
Ac supplemen migh p o ide a basis o de eloping a new
die a y s a egy o he p e en ion o RA.
M.A.R. pe o med he expe imen s and da a analysis. A.G.B,
J.G.F.B. and E.L. pe o med pa o he analysis. M.S.H. and
C.A. designed he s udy and p epa ed he manusc ip . All au ho s
ead and app o ed he inal con en o he manusc ip .
This s udy was suppo ed by esea ch g an s AGL 2011-26949
(Minis e io de Econom´
ıa y Compe i i idad, ISCIII, FEDER) and
P-10AGR-6609 (Jun a de Andaluc´
ıa). The au ho s g a e ully ac-
knowledge he assis ance o Cen e o Technology and Inno a ion
Resea ch, Uni e si y o Se ille (CITIUS).
The au ho s ha e decla ed no con lic s o in e es .
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RESULTADOS/DISCUSIÓN GENERAL

Resul ados/Discusión gene al
99
The ocus o he chap e I has been o demons a e he po en ial p o ec i e ole o a
die elabo a ed wi h EVOO in he p e en ion and de elopmen o in lamma o y a h i is and
join damage in a mu ine CIA-induced expe imen al a h i is model. This model is commonly
used o in es iga e mechanisms ele an o RA as well as new an ia h i ic ea men s
(Fe andiz, Maicas e al. 2008). CIA induc ion esul ed in he de elopmen o a p onounced
syno i is associa ed wi h an au oimmune esponse agains ca ilage and p oduc ion o ma ix
deg ading enzymes accompanying ca ilage deg ada ion and bone e osions (Schu ge s, Billiau
e al. 2011). The esul s o he p esen wo k clea ly indica e, o he i s ime, ha EVOO, as he
lipid componen o he die , e ec i ely exhibi ed p e en i e and he apeu ic e ec s in he
de elopmen o in lamma o y a h i is and join damage in CIA a h i ic mice in compa ison wi h
hose CIA mice ed wi h SO (Chap e I. Figu e 1 A and B). This e ec was well co ela ed o an
imp o ed a h i is sco e, a mino in lamma o y cells in il a ion in o a icula issues, educed
exuda ion in o he syno ial space, syno ial hype plasia and ca ilage e osion (Chap e I. Figu e
2).
The pa hogenesis o RA is closely ela ed o an imbalance o cy okine ne wo k
con ibu ing o he de elopmen and p og ession o bo h CIA and RA (Koma su and Takayanagi
2012). Ac ually, o e exp ession o p o-in lamma o y cy okines, such as IL-1β, TNF-α and IL-17
may ac i a e os eoclas s and mac ophages and also ec ui leukocy es in in lamed join s. The
in il a ed leukocy es p oduce cy okines, chemokines and issue des uc i e enzymes leading o
ch onic in lamma ion, ca ilage damage and bone e osion. Besides, IL-17 is able o induce he
elease o IL-8, IL-6 and plays a ema kable ole in he addi i e/syne gis ic e ec s induced by
TNF-α and IL-1β (Jeong, Kim e al. 2004). Impo an ly, a ge ing IL-17 has demons a ed
he apeu ic e ec s in animal models o in lamma o y and au oimmune diseases, including RA
(Miossec and Kolls 2012). Cy okines also up- egula e MMPs exp ession by chond ocy es and
syno iocy es a he pannus ca ilage junc ion leading o ca ilage deg ada ion (Fossiez, Djossou
e al. 1996, Azizi, Jadidi-Nia agh e al. 2013, Noack and Miossec 2014). The e o e, we examined
TNF-α, IL-1ß and IL-17 le els in paw homogena es om CIA animals in compa ison wi h naï e
con ol g oup. Ou esul s indica e ha animals ed wi h EVOO die showed a signi ican
educ ion in all p o in lamma o y cy okines le els. Consequen ly, EVOO in ake would con ibu e
o educe he in lamma o y esponse and join damage by supp essing he p oduc ion o he
key Th17 pola isa ion cy okines, TNF- α, and IL-1β, in CIA a h i ic mice (Chap e I. Figu e 4).
COMP, is a ma ix p o ein wi h a g ea po en ial as a biological ma ke o ca ilage me abolism
in a h i is (Saxne and Heinega d 1992). Inc eased agmen s o COMP ha e been epo ed in
pa ien s wi h os eoa h i is, RA and join inju y; hus, he moni o ing o COMP le els in syno ial
Resul ados/Discusión gene al
100
luid o se um has been sugges ed o be a help ul me hod o assessing he p esence and
p og ession o he in lamma o y disease. In his sense, se um COMP le els a e educed in RA
pa ien s in emission (Saxne and Heinega d 1992, Lai, Yu e al. 2012). In addi ion, COMP is a
pu a i e subs a e o MMPs. Pa icula ly, MMP-3, is a p o einase sec e ed by syno ial
ib oblas s and chond ocy es. I s ac i i y esul s in deg ada ion o agg ecan co e p o ein,
ca ilage link p o ein, ib onec in, and collagen ypes IV, VII, IX, and XI. MMP-3 is p esen in RA
syno ial luid and o e exp essed in heuma oid syno ium and hen, associa ed wi h highe join
damage in RA (Ally, Hodkinson e al. 2013). Likewise, se um MMP-3 le el is sugges ed as a
p edic o o join des uc ion in ea ly RA o es ablished RA and should be used in associa ion
wi h usual in lamma o y ma ke s o ollow he apy e iciency (Dena ie, Cons an e al. 2014).
Ou da a a e in ag eemen wi h abo e s udies and showed ha high se um COMP and MMP-3
le els we e associa ed wi h disease ac i i y and join p og ession in CIA mice, by con as he
p oduc ion o bo h ca ilage and syno ial bioma ke s we e signi ican ly inhibi ed by die a y
EVOO ea men in CIA mice (Chap e I. Figu e 3).
Abno mal signalling pa hways play an impo an ole in he in lamma o y p ocess and
can lead o dys egula ion o he in lamma o y esponse being c ucial in RA pa hogenesis. Signal
ansduc ion pa hways closely in ol ed in in lamma ion included he MAPKs, JAK-STAT and NF-
κB pa hways (Mo el and Be enbaum 2004). NF-κB is a c ucial ansc ip ional ac i a o o he
exp ession o mul iple p oin lamma o y genes in he mic oen i onmen o he a h i ic join s,
including TNF-α, IL-1β, IL-6 and IL-17, ha exe hei in luences on bo h os eoclas s and
os eoblas s di e en ia ion and he eby con ibu ing o p og essi e join des uc ion in RA
pa ien s and CIA mice (Okamo o, Yoshio e al. 2010, Moline o, Cub e e al. 2012) as men ioned
abo e. In ac , se e al an i- heuma ic d ugs, cu en ly in clinical use, inhibi NF-κB ac i a ion
(Hwang, Noh e al. 2013). In e es ingly, in a-a icula o sys emic blockade o NF-κB signalling
is an e ec i e a ge in he ea men o a h i is in animal models o RA (Min, Yan e al. 2015).
Taken oge he , hese s udies con i m he impo ance o NF-κB ac i a ion in he de elopmen
o RA. Consequen ly, NF-kB pa hway supp ession could be a no el s a egy o delaying he
p og ess o RA. Ou esul s sugges ed ha die a y EVOO ea men supp essed NF-κB ac i a ion
(Chap e I. Figu e 8), down- egula ing p o-in lamma o y cy okines and MMP-3 exp ession hus
minimizing join des uc ion in CIA-induced a h i ic mice. MAPK amily membe s, including p38
kinases, ERKs 1 and 2, and JNKs, play c i ical oles in many impo an cell p ocesses, including
cell di ision, di e en ia ion, in lamma ion, and apop osis. They a e especially d awing a en ion
in RA because o he in ol emen in egula ing bo h cy okine p oduc ion and cy okine ac ion
(C iado, Risco e al. 2014, Li, Li e al. 2014). In ac , JNK MAPK egula es MMPs p oduc ion by
Resul ados/Discusión gene al
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syno ial ib oblas s and d i es os eoclas di e en ia ion in RA (Han, Boyle e al. 2001) and p38
MAPK egula es MMP-3 induc ion in ib oblas s (Suzuki, Te suka e al. 2000) and os eoclas
di e en ia ion (Ma sumo o, Sudo e al. 2000). Mo eo e , MAPKs phospho yla e he janus kinase
signal ansduce and ac i a o o ansc ip ion (JAK-STAT) impo an in p oin lamma o y
cy okine-media ed signalling pa hway leading o STAT-3 ac i a ion (Aa onson and Ho a h
2002). STAT-3 has been desc ibed as a c i ical ansc ip ion ac o in ol ed in Th17 cell
di e en ia ion (Pa k, Lim e al. 2013). In his line, STAT-3 o e exp ession has been also de ec ed
in syno ial memb anes om RA pa ien s and i has been epo ed o con ibu e o he ch onici y
o in lamma ion in a mu ine zymosan-induced a h i is model (de Hooge, an de Loo e al. 2004).
Recen ly, STA-21, a p omising STAT-3 inhibi o has shown an ia h i ic e ec s in in e leukin-1
ecep o an agonis -knockou (IL-1Ra-KO) mice, an animal model o RA (Pa k, Kwok e al. 2014).
Ou s udy showed ha p38 and JNK MAPKs phospho yla ions we e inc eased in SO-CIA mice.
Simila ly, STAT-3 o e exp ession was also e idenced in RA syno ium o SO-CIA mice and was
posi i ely ela ed o he se e i y o syno i is. Whe eas EVOO die in ake educed signi ican ly
bo h MAPKs and STAT-3 ac i a ion a ansc ip ional le el (Chap e I. Figu es 5 and 7).
Al oge he , ou esul s sugges ha die a y EVOO may ep ess IL-17 p oduc ion in e e ing
nega i ely wi h JNK and p-38 MAPKs and STAT-3 signalling pa hways. HO-1 ac i i y ca abolises
heme o ca bon monoxide, i on and bili e din, he la e being educed o bili ubin by bili e din
educ ase. N 2 plays a cen al ole o exp ession o HO-1. In basal condi ions, N 2 is
seques e ed in he cy oplasm by Kelch like ECH-associa ed p o ein1 (Keap1) and deg aded by
he ubiqui in dependen 26S p o easome sys em. Unde ac i a ion, N 2 eleased om Keap1
inhibi ion, ansloca e o he nucleus, he e odime izes wi h Ma , and binds an ioxidan esponse
elemen s (AREs) loca ed in he p omo e egions o many de oxi ying/an ioxidan genes,
including HO-1 (Bang, Kim e al. 2012). In in lamma o y and immune condi ions he exp ession
o his p o ein could be pa o an adap a i e mechanism o limi cy o oxici y ia se e al
mechanisms including sca enging o eac i e oxygen o ni ogen species, egula ion o cell
p oli e a ion and p e en ion o apop osis (Fe andiz, Maicas e al. 2008). Likewise, i has been
epo ed ha HO-1 de iciency in mice esul s in a ch onic in lamma o y s a e (Minamino,
Ch is ou e al. 2001). Ou da a showed ha HO-1 could ep esen a po en ial molecula a ge
suscep ible o EVOO modula ion, which has no been demons a ed p e iously, since die a y
EVOO ea men s ongly augmen ed N 2 and HO-1 exp ession con e ing a ole o HO-1 in he
bene icial e ec s o EVOO in his mu ine model o ch onic in lamma ion (Chap e I. Figu e 6).
Since ou esul s show ha EVOO die was mo e e ec i e in educing a h i is se e i y
in compa ison wi h SO die , we could sugges ha he esponsibili y o such bene icial
Resul ados/Discusión gene al
102
p ope ies could o be assigned o bo h an adequa e a y acid p o ile o EVOO and he p esence
o a high p opo ion o phenolic compounds. In his sense, a ecen epo showed ha
oleu opein aglycone, a polyphenol ound in oli e oil, exe ed an an i-in lamma o y e ec and
amelio a ed he join damage associa ed wi h CIA (Impellizze i, Esposi o e al. 2011). In addi ion,
hese imp o ed e ec s obse ed could be due o a possible syne gis ic e ec among EVOO
cons i uen s, since i is no clea whe he all he possible bene icial mechanisms ac
independen ly o each o he o whe he hey ha e a syne gis ic o compe i i e ac ion (Lopez-
Mi anda, Pe ez-Jimenez e al. 2010, de la Las a Rome o 2011).
In he chap e II, we elucida ed he e ec s o o al PE om EVOO ea men in mu ine
CIA and s udy, he biochemical ou es and signalling pa hway in ol ed. Acco ding o ou esul s,
we ha e demons a ed ha o al adminis a ion o PE is able o down- egula e he a h i ic
p ocess in he CIA model o RA (Chap e II. Figu e 1 A and B). PE showed an i-in lamma o y
e ec s wi h educ ions in join oedema and mig a ion o in lamma o y cells. In addi ion, PE
p o ec ed join s agains ca ilage al e a ions and bone e osion (Chap e II. Figu e2). The
p og ession o a h i is is associa ed wi h sus ained p oduc ion o p oin lamma o y cy okines
(Go onzy and Weyand 2009). We ha e shown in he CIA model ha PE con ols he local le els
o hese cy okines d i ing he p oduc ion o di e en in lamma o y and ca abolic media o s
(Chap e II. Figu e 3). These indings a e in ag eemen wi h ou s p e ious esul s e alua ed in
CIA mice ed wi h die a y EVOO.
Pa icula ly, p oin lamma o y cy okines induce u he cy okines, chemokines,
eicosanoids and eac i e oxygen species ampli ying he in lamma o y esponse (Ab amson and
Amin 2002). The e o e, COX-2 and mPGES-1, enzymes esponsible o he o e p oduc ion o
PGE2 in in lamma ion, a e up- egula ed (Laza us, Kuba a e al. 2002) con ibu ing o he
p og ession o RA h ough EP4 ecep o ac i a ion (McCoy, Wicks e al. 2002). We ha e shown
ha PE educes PGE
2
le els (Chap e II. Figu e 3), which would be dependen on he down-
egula ion o COX-2 and mPGES-1 exp ession in he join (Chap e II. Figu e 4 A and B). Cy okines
also induce he exp ession o ma ix me allop o einases a he pannus ca ilage junc ion leading
o ca ilage deg ada ion (Okada, Nagase e al. 1987, Ribbens, Ma in y Po as e al. 2002). In
addi ion, TNFα s imula es os eoclas ogenesis (Cenci, Wei zmann e al. 2000), supp esses he
ec ui men o os eoblas s and inhibi s he exp ession o ma ix genes (Nanes 2003) whe eas IL-
6 inc eases os eoclas numbe s in abecula bone (Jilka, Hangoc e al. 1992). The e o e,
inhibi ion o p oin lamma o y cy okine p oduc ion by PE would esul in he educ ion o he
in lamma o y esponse and issue damage. MAPK egula e he syn hesis o chemokines,
cy okines, adhesion molecules and PGs in ol ed in in lamma ion. In addi ion, hey media e he
Resumen/ Abs ac
114
many o he bene icial e ec s o EVOO in ake a e due o i s mino highly bioac i e componen s
(abou 1–2 % o oil weigh ) (Ala con de la Las a e al. 2001)
Among hem, phenolic compounds such as hyd oxy y osol, y osol and oleu opein ha e
shown an i-in lamma o y and an ioxidan e ec s (Oma e al. 2010). Cu en expe imen al
s udies suppo a bene icial ole o polyphenols om EVOO in se e al in lamma o y diseases,
including RA (Ma inez-Dominguez, de la Pue a, and Ruiz-Gu ie ez 2001; Impellizze i e al.
2011; Gong e al. 2009). Al hough EVOO has demons a ed an i-in lamma o y e ec s, i has no
epo ed so much e idence o i s possible immunomodula o y e ec s.
The e o e, he objec i es o his hesis we e:
1. To de e mina e he possible p o ec i e e ec o die a y ex a i gin oli e oil (EVOO)
in collagen-induced a h i is (CIA) in DBA 1/J mice, an expe imen al model o RA and
explo e he biochemical ou es and possibly in acellula signalling pa hways.
2. To e alua e he o al polyphenolic ex ac om EVOO ea men in mu ine collagen-
induced a h i is and s udy he biochemical ou es and signalling pa hway in ol ed.
3. To in es iga e he e ec s o hyd oxy y osol (HTy) o hyd oxy y osol ace a e (HTy-
Ac), polyphenolics compounds om EVOO en iched-die s in expe imen al a h i is
model in mice and elucida e he molecula mechanisms and signalling pa hways
in ol ed.
Resul s and Discussion
1. Die a y ex a- i gin oli e oil p e en s in lamma o y esponse and ca ilage ma ix
deg ada ion in mu ine collagen‑induced a h i is. (Rosillo e al. Eu J Nu . 2015 In
p ess)
Th ee-week-old male DBA-1/J mice we e andomized in o ou expe imen al g oups: (1)
Sham sun lowe die (SO-Sham) g oup ecei ed a die elabo a ed wi h a ma ke able sun lowe
oil; (2) CIA sun lowe die (SO-CIA) g oup ecei ed a die elabo a ed wi h a ma ke able sun lowe
oil; (3) Sham EVOO die (EVOO-Sham) g oup we e ed wi h a die made wi h a ma ke able EVOO
picual a ie y and (4) CIA EVOO die (EVOO-CIA) g oup we e ed wi h a die made wi h a
ma ke able EVOO picual a ie y. A e 6 weeks, a h i is was induced by ype II collagen.

Resumen/Abs ac
115
Expe imen s ollowed a p o ocol app o ed by he Animal E hics Commi ee o he Uni e si y o
Se ille, and all expe imen s we e in acco dance wi h he ecommenda ions o he Eu opean
Union ega ding animal expe imen a ion (Di ec i e o he Eu opean Counsel 2010/630/EU)
Ou esul s e ealed, ha EVOO, as he lipid componen o he die , e ec i ely exhibi ed
p e en i e and he apeu ic e ec s in he de elopmen o in lamma o y a h i is and join
damage in CIA a h i ic mice in compa ison wi h hose CIA mice ed wi h SO. This e ec was
co ela ed o an imp o ed a h i is sco e, a mino in lamma o y cells in il a ion in o a icula
issues, educed exuda ion in o he syno ial space, syno ial hype plasia and ca ilage e osion.
O e exp ession o p o-in lamma o y cy okines, such as IL-1β, TNF-α and IL-17 may ac i a e
os eoclas s and mac ophages and ec ui leukocy es in in lamed join s. Besides, i is well-known
ha IL-17 is able o induce he elease o IL-8 and IL-6, and plays a ema kable ole in he
addi i e/syne gis ic e ec s induced by TNF-α and IL-1β (Jeong e al. 2004). Ou esul s indica e
ha animals ed wi h EVOO die showed a signi ican educ ion in IL-1β, TNF-α and IL-17 p o-
in lamma o y cy okines le els in paw homogena es.
Ca ilage oligome ic ma ix p o ein (COMP) is a ma ix p o ein wi h a g ea po en ial as
a biological ma ke o ca ilage me abolism in a h i is (Saxne and Heinega d 1992). In addi ion,
COMP is a pu a i e subs a e o me allop o einases (MMPs). Pa icula ly, MMP-3 is a
p o einase sec e ed by syno ial ib oblas s and chond ocy es and i s ac i i y esul s in
deg ada ion o agg ecan co e p o ein, ca ilage link p o ein, ib onec in and collagen. Ou da a
showed ha he p oduc ion o bo h ca ilage (COMP) and syno ial (MMP-3) bioma ke s was
signi ican ly inhibi ed by die a y EVOO ea men in CIA mice.
Abno mal signalling pa hways play an impo an ole in he in lamma o y p ocess and
can lead o a dys egula ion o he in lamma o y esponse being c ucial in RA pa hogenesis.
Nuclea ac o κB (NF-κB) is a c ucial ansc ip ional ac i a o o he exp ession o mul iple p o-
in lamma o y genes in ol ed in he mic oen i onmen o he a h i ic join s, playing an
impo an ole in he de elopmen o RA. (Mo el and Be enbaum 2004; Okamo o e al. 2010).
Ou esul s sugges ed ha die a y EVOO ea men supp essed NF-κB ac i a ion in CIA-induced
a h i ic mice. Simila ly, he mi ogen-ac i a ed p o ein kinase (MAPK) amily also plays c i ical
oles in RA pa hogenesis (Han e al. 2001; Suzuki e al. 2000) egula ing cy okine p oduc ion,
and ac i a ing he janus kinase-signal ansduce and ac i a o o ansc ip ion (JAK-STAT)
signalling pa hway h ough STAT-3 phospho yla ion (Aa onson and Ho a h 2002). Ou esul s
demons a ed ha EVOO die in ake educed signi ican ly bo h MAPKs and STAT-3 ac i a ion.
On he o he hand, nuclea ac o E2- ela ed ac o 2 (N 2) plays a cen al ole o exp ession
Resumen/ Abs ac
116
o heme oxigenase 1 (HO-1), an ioxidan enzyme. Ou da a showed ha HO-1 could ep esen a
po en ial molecula a ge suscep ible o EVOO modula ion, since die a y EVOO ea men
s ongly augmen ed N 2 and HO-1 p o ein exp ession con e ing a ole o HO-1 in he bene icial
e ec s o EVOO in his mu ine model o ch onic in lamma ion.
Al oge he , ou esul s con i m, o he i s ime, ha EVOO in ake d ama ically
a enua ed he p og ession and se e i y o a h i is in CIA DBA/1 J mice h ough N 2/HO-1
up egula ion and NF-κB, MAPKs and JAK-STAT signalling pa hway inhibi ion, dec easing he
in lamma o y cascade induced by CIA.
2. An i-in lamma o y and join p o ec i e e ec s o ex a- i gin oli e-oil polyphenol
ex ac in expe imen al a h i is. (Rosillo e al. Pha macol Res. 2012;66(3):235-42.)
CIA was induced in nine-weeks-old male DBA-1/J mice. On day 29, animals we e
andomized in ou g oups o mice: naï e g oup (NA), CIA g oup (CIA) and wo ea men g oups:
CIA mice wi h polyphenolic ex ac (PE) ea men (PE 100 and 200 mg/kg, o ally, once a day
om days 29 o 41).
In his s udy, we ha e demons a ed ha o al adminis a ion o PE was able o down-
egula e he a h i ic p ocess in he CIA model o RA. PE showed an i-in lamma o y e ec s wi h
educ ions in join edema and mig a ion o in lamma o y cells. In addi ion, PE p o ec ed join s
agains ca ilage al e a ions and bone e osion. The p og ession o a h i is is associa ed wi h
sus ained p oduc ion o p o-in lamma o y cy okines such as TNF-α, IL-1β and IL-6 (Go onzy and
Weyand 2009). Ou da a shown ha PE ea men con ols he local le els o hese cy okines,
d i ing he p oduc ion o di e en in lamma o y and ca abolic media o s in CIA model.
In his sense, COX-2 and mic osomal p os aglandin E syn hase-1 (mPGES-1), enzymes
esponsible o he o e p oduc ion o PGE
2
in in lamma ion, a e d ama ically up- egula ed
(Laza us e al. 2002) con ibu ing o he p og ession o RA h ough EP
4
ecep o ac i a ion
(McCoy, Wicks, and Audoly 2002). Ou da a sugges ha PE educes PGE
2
le els, which would be
dependen on he down- egula ion o COX-2 and mPGES-1 p o ein exp ession in he join .
In addi ion, PE ea men inhibi s he phospho ila ion o JNK and p38 MAPK and
ansc ip ion ac o STAT-3. Simila ly, PE addi ion dec eases NF-κB nuclea ansloca ion leading
o he down- egula ion o he a h i ic p ocess.
Resumen/Abs ac
117
In summa y, ou s udy has demons a ed, o he i s ime, he an i-in lamma o y and
join p o ec i e e ec s o PE om EVOO in a CIA-induced expe imen al model, which would be
ela ed o he inhibi ion o ele an signalling pa hways such as NF-κB, JAK/STAT and MAPKs
con olling he p oduc ion o in lamma o y media o s.
3. P e en i e e ec s o die a y hyd oxy y osol ace a e, an ex a i gin oli e oil
polyphenol in mu ine collagen-induced a h i is. (Rosillo e al. Mol Nu Food Res. 2015.
In p ess)
Th ee-weeks-old male DBA-1/J mice, we e andomized in o ou expe imen al g oups
du ing all expe imen al pe iod: (i) naï e g oup ecei ed a s anda d die (SD), (ii) CIA g oup
ecei ed a SD, (iii) CIA-HTy g oup ecei ed a 0.05% HTy die and (i ) CIA-HTy-Ac g oup ecei ed
a 0.05% HTy-Ac die . A e 6 weeks, a h i is was induced by ype II collagen.
HTy-Ac was desc ibed o he i s ime in oli e oil by B enes e al. (B enes e al. 1999)
and is ound in mos Spanish i gin oli e oils. Mo eo e , ecen ly, i was epo ed ha HTy-Ac is
mo e soluble in he lipophilic phases han HTy, due o he p esence o he es e g oup. Thus,
his inc eased lipophilici y sugges s ha HTy-Ac is be e abso bed ac oss in es inal epi helial
cell monolaye s han ee HTy (Rubio e al. 2012).
Ou indings ha e e ealed ha die a y HTy-Ac supplemen a ion could imp o e he
a h i is sco e which was co ela ed wi h a mino mig a ion o in lamma o y cells in o a icula
issues in addi ion o a ma ked educ ion o join edema, syno ial hype plasia and ca ilage
e osion in compa ison wi h hose animals ed wi h s anda d die (SD) and hyd oxy y osol (HTy)
en iched die s.
CIA pa hogenesis is cha ac e ized by he gene a ion o an i-CII an i-bodies (Nandakuma
e al. 2003). All iso ypes (IgG1, IgG2a and IgG2b) o an i-CII an ibodies we e a h i ogenic, wi h
he IgG1 and IgG2b iso ypes as he domina ing a h i ogenic an ibodies (Nandakuma e al.
2003). Ou esul s demons a ed ha die a y HTy-Ac supplemen a ion signi ican ly dec eased
se um le els o IgG1-and IgG2a-an i-CII an ibodies in CIA mice. These esul s sugges a po en ial
ole o HTy-Ac in egula ing B cell esponses, which may be in pa , esponsible o i s an i-
a h i ic e ec s.
Ou esul s shown ha a h i ic mice ed wi h HTy-Ac en iched die had dec eased
se um COMP and MMP-3 le els, as well as, p o-in lamma o y cy okines such as IL-1β, IL-6, IFN-
γ, IL-17 and TNF-α le els in paw homogena e in compa ison wi h CIA mice ed wi h SD and HTy
Resumen/ Abs ac
118
en iched die . Mo eo e , he ac i a ion o JAK/STAT, MAPKs and NF-κB pa hways we e
d as ically amelio a ed whe eas N 2 and HO-1 p o ein exp essions we e signi ican ly up-
egula ed in hose mice ed wi h HTy-Ac.
As a conclusion, ou s udy e eals, o he i s ime, he an i-in lamma o y e ec s o
die a y HTy-Ac in he CIA model. The mechanisms unde lying hese p o ec i e e ec s could be
ela ed o he egula ion o B cell esponses, ac i a ion o he N 2/HO-1 signalling pa hway and
he inhibi ion o ele an signalling pa hways such as JAK-STAT, MAPKs and NF-κB con olling
he p oduc ion o in lamma o y media o s.
Conclusion
Al oge he , ou esul s sugges ha EVOO exe s p e en i e e ec s in he de elopmen
o expe imen al RA, playing i s mul iple mino componen s a key ole in hese heal hy bene i s.
Thus, EVOO may be conside ed as suppo i e nu i ional he apy o RA pa ien s as well as i s
poliphenolic ac ion migh p o ide an a ac i e nu aceu ical complemen in managemen o
RA, mainly when he e ec s o inexpensi e, side e ec - ee he apies based on die a y EVOO
migh suppose an imp o emen in public heal h on he p e en ion o ch onic pa hologies wi h
high p e alence in he popula ion.
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