Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in [ MOLECULAR NUTRITION & FOOD RESEARCH] on [18 September 2015], available at: https://doi.org/[https://doi.org/10.1002/mnfr.201500304].” For Peer Review Preventive effects of dietary hydrox ytyrosol acetate, an extra virgin olive oil polyphenol in murine collageninduced arthritis Journal: Molecular Nutrition and Food Research Manuscript ID: mnfr.201500304.R2 Wiley - Manuscript type: Research Article Date Submitted by the Author: n/a Complete List of Authors: Rosillo, Maria Angeles; University of Seville, Pharmacology Sánchez-Hidalgo, Marina; University of Seville, Pharmacology González-Benjumea, Alejandro; University of Seville, Organic Chemistry Fernández-Bolaños, José G.; University of Seville, Organic Chemistry Lubberts, Erik; Erasmus Medical Center, Rheumatology Alarcón-de-la-Lastra, Catalina; University of Seville, Pharmacology Keywords: CIA, EVOO, Hydroxytyrosol acetate, Inflammation, Rheumatoid arthritis Wiley-VCH Molecular Nutrition and Food Research For Peer Review Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis María Angeles Rosillo 1 , Marina Sánchez-Hidalgo 1 , Alejandro González- Benjumea 2 , José G. Fernández-Bolaños 2 , Erik Lubberts 3 , Catalina Alarcón-de-la- Lastra 1, * 1 Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain 2 Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Spain 3 Department of Rheumatology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands * Corresponding author: Dr. Catalina Alarcón de la Lastra Department of Pharmacology. Faculty of Pharmacy. University of Seville, Spain. Profesor García González, 2. 41012 Seville Telephone: +34 954 559877 Fax: + 34 954 55 6074 [email protected]s Abbreviations (COMP) cartilage olimeric matrix protein, (COX-2) cyclooxygenase-2, (ERK) extracellular signalregulated kinases, (EVOO) extra virgin olive oil, (H&E) hematoxylin and eosin, (HO-1) heme oxygenase-1, (HTy) hydroxytyrosol, (HTy-Ac) Hydroxytyrosol acetate, (JAK/STAT) Janus kinasesignal transducer and activator of transcription, (JNK) c-Jun N-terminal kinases, (MAPKs) mitogen-activated protein kinases, (MMPs) metalloproteinases, (MMP-3) metalloproteinase-3, (mPGES-1) prostaglandin E synthase-1, (NF-κB) nuclear transcription factor-kappa B, (Nrf2) nuclear factor E2-related factor 2, (RA) rheumatoid arthritis, (SD) standard diet, (STAT-3) signal transducer and activator of transcription (TNF-α) tumor necrosis factor α Keywords CIA, EVOO, Hydroxytyrosol acetate, Inflammation, Rheumatoid arthritis Page 1 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Abstract Scope: Hydroxytyrosol acetate (HTy-Ac), an extra virgin olive oil (EVOO) polyphenol, has recently exhibited antioxidant and anti-inflammatory effects on LPS-stimulated macrophages and ulcerative colitis. This study was designed to evaluate dietary HTy-Ac supplementation effects on collagen-induced arthritis (CIA) in mice. Methods and results: DBA-1/J mice were fed from weaning with 0.05% HTy-Ac. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrificed 42 days after first immunization. Blood was recollected and paws were histological and biochemically processed. HTy-Ac diet significantly prevented arthritis development and decreased serum IgG1 and IgG2a, cartilage olimeric matrix protein (COMP) and metalloproteinase-3 (MMP-3) levels, as well as, pro-inflammatory cytokines levels (TNF-α, IFN-γ, IL-1β, IL-6 and IL-17A). The activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-κB) pathways were drastically ameliorated whereas nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions were significantly up-regulated in those mice fed with HTy-Ac. Conclusion: HTy-Ac improved the oxidative events and returned pro-inflammatory proteins expression to basal levels probably through JAK/STAT, MAPKs and NF-κB pathways. HTy-Ac supplement might provide a basis for developing a new dietary strategy for the prevention of rheumatoid arthritis. Page 2 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 1. Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by inflammation of multiple joints and destruction of cartilage and bone. The earliest event is the development of systemic autoimmunity by exogenous and autologous antigens and thus autoantibodies and later high levels of cytokines can be detected years before clinical symptom [1, 2]. At a joint level, there are synovial hyperplasia and massive infiltration of immune cells, including CD4 + T-cells, B-cells, natural killer cells, macrophages, dendritic cells, neutrophils and mast cells. Although the cause of RA remains unclear, increasing evidences indicate that proinflammatory cytokines such as tumor necrosis factor α (TNFα), IL-1β, IL-6, IFN-γ and metalloproteinases (MMPs) produced from RA synovium play an important role in the erosion of articular cartilage and subchondral bones [3, 4]. The process of gene expression of these pro-inflammatory mediators involves multiple signal transduction pathways including mitogen-activated protein kinases (MAPKs) which comprises extracellular signal-regulated kinases (ERK1/2 or p42/p44), c-Jun N-terminal kinases (JNK)1/2/3 and p38, and the nuclear transcription factor-kappa B (NF-κB) which are activated in the synovium of patients with RA [5, 6]. In particular, NF-κB plays an important role in MMPs induction and also regulates a wide range of genes that contribute to inflammation, such IL-1β, TNFα, IL-6, chemokines and microsomal prostaglandin E synthase-1 (mPGES-1), an efficient downstream enzyme co-localized and functionally coupled with the inducible enzymes cyclooxygenase-2 (COX-2). Both, COX-2 and mPGES-1, are up-regulated and responsible for the overproduction of prostaglandin E 2 (PGE 2 ) which may affect joint integrity through EP 4 receptor activation [7]. The signal transducer and activator of transcription (STAT)-3 is another critical transcription factor inflammatory pathway activated in response to cytokines involved in the pathogenesis of RA by steering the abnormal activation, automaticity, and prolonged survival of synovial cells. Specially, overexpression of STAT-3 has been reported in synovial membranes from RA patients correlating with paired serum IL-6 [8]. Likewise, nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor orchestrator of the induction of several antioxidant enzymes, such as heme oxygenase (HO)-1. The activation of HO-1 in inflammatory conditions could be part of an adaptive mechanism to limit cytotoxicity. In fact, it has been reported that HO-1 deficiency in mice results in a chronic inflammatory state [9]. Despite significant advances in therapies for the treatment of many autoimmune diseases such as RA, a persistent unmet need still exists for more effective, durable, and convenient treatment options. In this sense, the interest by dietary supplements and nutraceuticals without undesirable effects that accompany the classical pharmacotherapy is growing. Current epidemiological and experimental studies support a beneficial role of dietary polyphenols in several inflammatory diseases, including RA. In this regard, we have previously demonstrated that oral administration of a phenolic extract from extra virgin olive oil (EVOO) was able to down-regulate the arthritic process in the collagen-induced arthritis (CIA) model of RA [10]. EVOO is rich in a variety of phenolic compounds, mainly constituted by secoiridoid derivatives of 2-(3,4-dihydroxyphenyl)ethanol (hydroxytyrosol, HTy) and of 2-(4-hydroxyphenyl)ethanol (tyrosol), and hydroxytyrosyl acetate (HTy-Ac), along with minor amounts of free HTy [11]. To date, HTy-Ac has shown protection effects against oxidative DNA damage in blood cells [12], as well as against iron-induced oxidative stress in human cervical cells (HeLa) [13]. In addition, a study from González-Correa et al., [14] showed a neuroprotective effect of HTy-Ac in a model of hypoxia–reoxygenation in rat brain slices, both in vitro and after oral administration. A greater antiplatelet aggregating activity than HTy has also been demonstrated [15]. More recently, we have showed that HTy-Ac, exerted an anti-inflammatory effect on acute ulcerative colitis. This effect involves a decrease in COX-2 and iNOS protein expression probably trough JNK MAPK and NF-κB signaling pathways [16]. In addition HTy-Ac was able to modulate inflammatory response in murine peritoneal macrophages [17]. Page 3 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Taken this background into account, the present study was designed to evaluate the effects of HTy-Ac dietary supplementation, in the arthritis model of CIA in mice. In addition to macroscopic and histological analyses, we have determined the effects of HTy-Ac on the production of inflammatory mediators. In order to gain a better insight into mechanisms of action, signaling pathways were also explored. 2. Material and Methods. To evaluate the beneficial effects of HTy-Ac on CIA model, DBA 1J mice were used. Mice were randomized in four experimental groups (10 animals per group): (1) naïve group, (2) Control group (CIA), (3) HTy diet group (CIA-HTy) and (4) HTy-AC diet group (CIA-HTy-Ac). After 6 weeks, arthritis was induced by type II collagen. Mice were sacrificed 42 days after first immunization. Blood was recollected and paws were histological and biochemically processed. ELISA, histopathological analysis and immunoblotting were performed as indicated in Supporting Information Material and Methods. 3. Results 3.1. Effects of dietary HTy-Ac on CIA-induced AR model. The development of arthritis was monitored until day 42. The time-course of arthritic score indicates that control CIA mice developed a progressive development of arthritis observed from day 34 (Fig. 1A). However, mice fed with HTy-Ac diet showed a significant delayed onset (p<0.05 and p<0.01 vs. CIA) and decreased the disease severity of CIA reducing disease incidence, number of involved paws, footpad thickness and clinical index from days 37 to 42. Surprisingly, HTy dietary did not modify the development of arthritis. These results suggested that dietary enriched with HTy-Ac not only could retard the development but it also may have a therapeutic effect on ongoing inflammatory arthritis. Representative photographs of hind paws from the different experimental animal groups are shown in figure 1B. In addition, H&E staining revealed that histological features of the joint from naïve animals were typical of normal structure with synovial membrane composed of synovial cells and collagen and a clear synovial space (Fig. 1C, a). On the contrary, joint from control CIA mice exhibited histological changes indicative of severe arthritis, characterized by an extensive infiltration of inflammatory cells into articular tissues, exudation into the synovial space, synovial hyperplasia and cartilage erosion (Fig. 1C, b). These histological features were less evident in CIA -HTy-Ac group (Fig. 1C, d). 3.2. HTy-Ac diet reduced levels of CII-specific antibodies. To determine the effect of HTy-Ac dietary on autoantibody production, serum was collected at day 42 and anti-CII specific IgG antibodies were measured. As shown in Figure 2A, bovine IgG1 and IgG2a levels were significantly lower in dietary HTy-Ac feed group in comparison with CIA control (p< 0.01 vs. CIA) and HTy feed group (p<0.01 and p<0.05 vs. HTy group). Similarly, mouse IgG1 and IgG2a levels were also significantly ameliorated in mice fed with HTy-Ac diet (p<0.01 and p<0.05 vs. CIA and p<0.05 vs. HTy group) 3.3. HTy-Ac diet decreased serum MMP-3 and COMP levels in CIA-induced RA. In the present study we determined serum MMP-3 levels, as synovial inflammatory biomarker, in CIA mice to investigate the potential beneficial effects of our nutritional therapeutic strategy. Circulating MMP-3 levels were markedly increased (p<0.01 vs. naïve) in serum from arthritic CIA control group (Fig. 2C). By contrast, a reduction in MMP-3 serum levels was observed in those arthritic animals feeding with HTy-Ac (p<0.05 vs. CIA). In addition, serum levels of COMP were significantly elevated in CIA animals when compared to naïve controls (p<0.001 vs. naïve). On the contrary, CIA-HTy-Ac animal group significantly decreased COMP levels in comparison with CIA group (p<0.001 vs. CIA) reaching levels comparable to those described in naïve animals (Fig. 2C); whereas dietary HTy treatment was ineffective. 3.4. Effects of dietary HTy-Ac on joint mediators. It has been reported that TNF-α, IL-1β, IL-6, IFN-γ and IL-17A are critical cytokines involved in the pathogenesis of RA [1]. To explore whether cytokines joint levels were paralleled to the disease severity of CIA, concentration of these cytokines were examined in paw homogenates Page 4 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review by ELISA. As shown in Figure 3, TNF-α, IL-1β, IL-6, IFN-γ and IL-17A levels were significantly increased in paw homogenates from CIA animals when compared with naïve mice (p<0.01 and p<0.01 vs. naïve) suggesting its relationship with the synovial tissue inflammation. Conversely, our results indicate that animals fed with HTy-Ac diet showed a significant reduction in all those pro-inflammatory cytokines production in comparison with CIA group (TNF-α: p<0.05, IL- 1β: p<0.001, IL-6: p<0.01, IFN-γ: p<0.01 and IL-17A: p<0.001 vs. CIA). 3.5. Effect of dietary HTy-Ac on COX-2 and mPGES-1 expression. COX-2 and mPGES1 expressions were determined by western blot in paw homogenate (Fig. 4A). Arthritic control animal group showed an overexpression of both these pro-inflammatory enzymes (COX-2: p<0.01 and mPGES1: p<0.001 vs. naïve) whereas HTy-Ac diet was able to reduce the protein expression levels of both them (p<0.01 vs. CIA) (Fig. 4B). By contrast, dietary HTy treatment failed to reduce significantly the protein expression levels of both COX-2 and mPGES1 in CIA mice. On the other hand, levels of PGE 2 were measured on the paw homogenate, HTy-Ac was capable to reduce the levels of these eicosanoid. (Fig. 4B) 3.6. Effect of dietary HTy-Ac on p-STAT-3 protein expression. STAT-3 has been described as a critical transcription factor involved in the pathogenesis of RA by steering the abnormal activation, automaticity, and prolonged survival of synovial cells. It has been also described that IL-6 active the JAK/STAT pathway and mainly culminates in the activation of the STAT-3 transcription factor [18]. We evaluated p-STAT-3 protein expression by western blot from hind paw homogenates. Statistical analysis revealed a significant p-STAT- 3 overexpression in CIA group when compared to the naïve control groups (p<0.001 vs. naïve) whereas nutritional therapy with HTy-Ac significantly suppressed STAT3 phosphorylation in arthritic CIA mice (p<0.01 vs. CIA), this suppression was also significant in comparison with HTy-CIA group (p<0.01 vs. CIA -HTy) (Fig. 5A). These results are in accordance with those obtained in the measurement of pro-inflammatory cytokines levels suggesting that dietary HTy-Ac may repress STAT-3 activation reducing IL-6 levels in CIA mice. 3.7. Dietary HTy-Ac induces Nrf2/HO-1 antioxidant pathway activation. The expression of the proteins HO-1 and Nrf2 were also evaluated in paw homogenates by western blotting. Our data show that HO-1 was significantly downregulated in CIA mice during the maintenance of chronic inflammation (p<0.001 vs. naïve); however, dietary HTy-Ac treatment induced a HO-1 overexpression in comparison with CIA arthritic control group (p<0.05 vs. CIA) (Fig. 5B). Nrf2 activation has been reported to play an important role in HO-1 expression. According to our results, Nrf2 expression was similarly reduced in those arthritic animals fed with SD diet (p<0.001 vs. naïve) whereas a significant increase in Nrf2 protein levels was observed in CIA-HTy-Ac animals (p<0.05 vs. CIA and p<0.05 vs. CIA-HTy) (Fig.5B). 3.8. Effect of dietary HTy-Ac on MAPKs signaling pathway. MAPKs play a key role inducing the pro-inflammatory gene expression which initiates inflammatory responses. We investigated the effect of dietary HTy-Ac on MAPKs (JNK and p38) signaling pathway activation in CIA mice. In the present study, phosphorylation of JNK, p38 and ERK (1/2) proteins increased significantly in cytosolic extracts from CIA mice paw homogenates. Nonetheless, the proteins expression of all phosphorylated MAPKs proteins, p- JNK, p-p38 and p-ERK (1/2) was significantly ameliorated after dietary HTy-Ac treatment (p<0.05 and p<0.01 vs. CIA) (Fig. 6A). Again, the HTy diet showed its ineffectiveness along the experimental period. 3.9. Effects of dietary HTy-Ac on NF-κB signaling pathway. We also investigated the effects of HTy-Ac on IkB-α degradation in cytoplasmic extracts from mice joints. As shown in figure 6B, IκB-α expression was significantly reduced in CIA-induced arthritis mice when compared with naïve mice (p < 0.001 vs. naïve). The IκB-α expression observed in CIA mice was consistent with an increase in IκB-α degradation thus allowing NF-κB translocation into the nucleus to bind specific DNA sequences leading to pro-inflammatory genes transcription. According to the results obtained, the effect observed in arthritic control group on IκB-α protein degradation was prevented by dietary HTy-Ac treatment. On the Page 5 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review contrary, the nuclear p65 protein levels were significantly increased in CIA group (p<0.001 vs. naïve) whereas dietary HTy-Ac treatment prevented the CIA-induced nuclear translocation level of p65 in paw homogenate in comparison with those arthritic animals fed with standard diet (p<0.01 vs. CIA) (Fig. 6B) avoiding the NF-kB-mediated transcriptional activation. HTy diet failed to modify IκB-α degradation and nuclear p65 translocation in CIA mice. 4. Discussion Our findings, have shown, for the first time, that dietary HTy-Ac enrichment was able to prevent and down-regulate the arthritic process in the CIA model of RA. This model is commonly used to investigate relevant pathogenic mechanisms of RA as well as new antiarthritic treatments [19]. HTy-Ac was described for the first time in olive oil by Brenes et al.[20] and is found in most Spanish virgin olive oils. Moreover, recently, it was reported by Mateos et al.[21] that HTy-Ac is more soluble in the lipophilic phases than HTy, due to the presence of the ester group, which was demonstrated in a Caco-2 cell model. Thus, this increased lipophilicity means that HTy-Ac is better absorbed across intestinal epithelial cell monolayers than free HTy [22]. CIA induction resulted in the development of a pronounced synovitis associated with cartilage degradation and bone erosion [23]. However, dietary HTy- Ac supplementation could improve the arthritis score which was correlated with a minor migration of inflammatory cells into articular tissues in addition to a marked reduction of joint edema, synovial hyperplasia and cartilage erosion in comparison with those animals fed with SD and HTy enriched diets. A role of humoral immunity in the pathogenesis of autoimmune arthritis is suggested by evidence derived from animal models as well as patients with RA. Antibody/antigen complexes are abundantly found in the joints of RA patients and are believed to play a role in triggering the joint inflammation [24]. CIA pathogenesis is characterized by the generation of anti-CII anti-bodies. All isotypes (IgG1, IgG2a and IgG2b) of anti-CII antibodies were arthritogenic, with the IgG1 and IgG2b isotypes as the dominating arthritogenic antibodies [25]. In the present study, dietary HTy-Ac supplementation significantly decreased serum levels of IgG1-and IgG2a- anti-CII antibodies in CIA mice. These results suggest a potential role for HTy-Ac in regulating B cell responses, which may be in part, responsible for its anti-arthritic effects. The development and progression of RA is closely related to an imbalance of cytokine network. In RA synovium, elevated levels of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-17 and INF-γ are produced by macrophages and synovial fibroblasts. These pro-inflammatory cytokines both directly and indirectly exert their effects through the production of additional pro-inflammatory cytokines, chemokines and MMPs at the pannus cartilage junction leading to cartilage degradation [26-28]. In addition, TNF-α stimulates osteoclastogenesis [29], suppresses the recruitment of osteoblasts and inhibit the expression of matrix genes [30], whereas IL-6 increases osteoclast numbers in trabecular bone [31]. Besides, IL-17 is a T cellderived cytokine able to induce the release of IL-8 and IL-6, and plays a considerable role in the additive/synergistic effects induced by TNF-α and IL-1β [32]. The present study showed that arthritic mice fed with HTy-Ac enriched diet had decreased IL- 1β, IL-6, IFN-γ, IL-17 and TNF-α levels in paw homogenate in comparison with CIA mice fed with SD and HTy enriched diet. These results indicate that dietary HTy-Ac exerts antiinflammatory activities by the blockage of IL-1β, IL-6, IFN-γ, IL-17 and TNF-α production. COMP, a prominent non-collagenous component of cartilage, accounts for approximately 1% of the wet weight of articular tissue and shows great potential as a biological marker of cartilage metabolism in arthritis [33]. Increased fragments of COMP have been reported in patients with osteoarthritis, RA, and joint injury, thus and the monitoring of COMP levels in synovial fluid or serum has been suggested to be a helpful method for assessing the presence and progression of arthritis. In fact, serum COMP is reduced in RA patients in remission[33]. In addition, COMP is a putative substrate for MMPs. In particular, MMP-3, is a proteinase secreted by synovial fibroblasts and chondrocytes. Its synthesis and activation is induced by various factors, including pro-inflammatory cytokines and Toll-like receptor ligand. Its activity Page 6 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review is associated with higher joint damage in RA [34] and CIA [4] and results in degradation of aggrecan core protein, cartilage link protein, fibronectin, and collagen types IV, VII, IX, and XI. Likewise, serum MMP-3 level is suggested as a predictor for joint destruction in early RA or established RA and should be used in association with usual inflammatory markers to follow therapy efficiency [35]. Our data are in agreement with above studies and showed that high serum COMP and MMP-3 levels were associated with disease activity and joint progression in CIA mice, by contrast the production of both cartilage and synovial biomarkers were significantly inhibited by the HTy-Ac enriched diet in CIA mice. COX-2 and mPGES-1, enzymes responsible for the overproduction of PGE 2 in inflammation, are up-regulated [36] contributing to the progression of RA through EP 4 receptor activation [7]. We have shown that dietary HTy-Ac supplementation reduced PGE2 levels which could be possibly due to decreased expression of both COX-2 and mPGES-1 in the joint. Therefore, regulation of these pro-inflammatory biomarkers by HTy-Ac could represent a potential molecular target susceptible to HTy-Ac modulation, which has not been demonstrated previously. Signal transduction pathways closely involved in inflammation include the MAPKs, JAK-STAT and NF-κB pathways [37, 38]. In fact, NF-κB nuclear transcription factor plays a pivotal role in the development and activation of Th-1 responses [39] and is responsible in addition to MAPKs for COX-2 up-regulation [40]. We investigated whether the activation of NF-κB signaling pathway was inhibited by HTy-Ac diet. Our data are in agreement with Sanchez-Fidalgo et al. that showed that HTy-Ac increased the inhibitory protein IkB-α and reduced p65 translocation, indicating that dietary HTy-Ac inhibited the NF-κB activation by blocking IκB-α degradation in dextran sulfate sodium -induced colitis in mice [16]. MAPK family members, including p38 kinases, ERKs 1 and 2 and JNKs, are involved in many important cell processes, mainly the regulation of the synthesis of chemokines, cytokines, adhesion molecules and PGs involved in RA [6, 41]. Besides JNK MAPK modulates MMPs production by synovial fibroblasts and drives osteoclast differentiation in RA [42]. In particular, p38 MAPK regulates MMP-3 induction in fibroblasts [43] and osteoclast differentiation [44]. Moreover, MAPKs phosphorylate the JAK-STAT important in pro-inflammatory cytokinemediated signaling pathways such as Th17 cell differentiation, leading to STAT-3 activation by phosphorylation on tyrosine residues resulting in the formation of STAT dimers that translocate into the nucleus to bind specific DNA sequences [45]. In this line, STAT-3 overexpression has been also detected in synovial membranes from RA patients and it has been reported to contribute to the chronicity of CIA induced arthritis model [4, 46]. Our findings are in concordance with above reports and showed that p38 and JNK MAPKs phosphorylation were increased in CIA control mice. Similarly, STAT-3 overexpression was also evidenced in RA synovium of control CIA mice and was positively related to the severity of synovitis, whereas dietary HTy-Ac supplementation reduced significantly both MAPKs and STAT-3 activation at transcriptional level. Collectively, our data suggest that dietary HTy-Ac may repress IL-17 production interfering negatively with JNK, p-38, p-ERK MAPKs and STAT-3 signaling pathways. Nrf2, is a redox-sensitive transcription factor and binds to antioxidant response elements (ARE) located in the promoter regions of many detoxifying/antioxidant genes, including HO-1 [47]. In inflammatory conditions, HO-1 expression protein could be part of an adaptive mechanism to limit cytotoxicity via several mechanisms including scavenging of reactive oxygen or nitrogen species, regulation of cell proliferation and prevention of apoptosis. Likewise, it has been reported that HO-1 deficiency in mice results in a chronic inflammatory state [9]. Thus, Nrf2 regulates redox status and plays key roles in cellular defense by enhancing the removal of reactive oxygen species [48]. Furthermore, it has been documented that deficiency of Nrf2 accelerates the effector phase of arthritis and aggravates joint disease [49]. In the present study, in concordance with Karatas et al. [50], expressions of Nrf2 and HO-1 were decreased in the arthritis group, by contrast dietary HTy-Ac could restore Nrf2 and HO-1 expressions Page 7 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 254x190mm (96 x 96 DPI) Page 14 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 254x190mm (96 x 96 DPI) Page 15 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 254x190mm (96 x 96 DPI) Page 16 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 254x190mm (96 x 96 DPI) Page 17 of 17 Wiley-VCH Molecular Nutrition and Food Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60