Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Resveratrol Alleviates Rheumatoid Arthritis via Reducing ROS and Inflammation, Inhibiting MAPK Signaling Pathways, and Suppressing Angiogenesis Guliang Yang,† Chia-Che Chang,*,§,∥,⊥,#,▽ Yiwen Yang,† Li Yuan,† Leishiyuan Xu,† Chi-Tang Ho,‡ and Shiming Li*,†,‡ †
Hubei Key Laboratory of EFGIR, Huanggang Normal University, Huanggang, Hubei 438000, China Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States § Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan ∥ Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan ⊥ The iEGG and Animal Biotechnology Research Center, National Chung Hsing University, Taichung 40227, Taiwan # Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan ▽ Department of Biotechnology, Asia University, Taichung 41354, Taiwan
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ABSTRACT: Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting joints and is featured by chronic synovial inflammation and angiogenesis. We employed a bovine type-II collagen (BIIC)-induced Sprague−Dawley rat arthritis model and an in vitro RA model based on interleukin (IL)-1β-stimulated rat synovial cells (RSC-364) to explore the preventive effect of resveratrol on RA and the underlying mechanisms. We found that resveratrol ameliorated BIIC-elicited synovitis and RA-related pathological hallmarks such as inflammatory cell infiltration and angiogenesis in the synovial tissue. Also, BIICstimulated rats displayed increased serum levels of proinflammatory cytokines and reactive oxygen species (ROS), as manifested by elevated serum malonaldehyde contents combined with reduced superoxide dismutase activity. It is noteworthy that resveratrol abolished BIIC-induced ROS and inflammation, confirming the antioxidative and anti-inflammatory actions of resveratrol in the context of RA. Furthermore, immunoblotting indicated that resveratrol downregulated the increase in the levels of hypoxia-inducible factor-1α (HIF-1α) and that of the activated phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in IL-1β-stimulated RSC-364 cells. Moreover, we observed that resveratrol-treated RSC-364 cells displayed both G0/G1 cell-cycle arrest and enhanced levels of apoptosis. Altogether, the present evidence established the preventive role of resveratrol in RA progression. Mechanistically, resveratrol inhibits MAPK signaling pathways, likely by reducing ROS accumulation, to suppress the inflammatory response and cell proliferation and to provoke cell apoptosis in the synovial tissue, along with mitigation of HIF-1α-mediated angiogenesis. Thus resveratrol appears to hold great potential for clinical translation as a novel RA therapeutic. KEYWORDS: rheumatoid arthritis (RA), resveratrol, ROS, angiogenesis, MAPK
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INTRODUCTION Rheumatoid arthritis (RA) is a systematic autoimmune disease characterized by chronic synovial inflammation along with synovial hyperplasia and invasion into surrounding cartilage and bone, leading to progressive damage to the knee joints.1 Angiogenesis is essential for the pathogenesis of inflammatory diseases, including RA. In RA development, angiogenesis in synovial tissues is required for sustaining chronic inflammatory state and synovial hyperplasia.2 Additionally, increased levels of reactive oxygen species (ROS) at the site of inflammation are fundamental to the progression of joint lesions.3,4 Notably, ROS activates mitogen-activated protein kinase (MAPK) signaling pathways, which are responsible for regulating the production of proinflammatory cytokines and downstream events culminating in joint inflammation and destruction.5,6 Although current therapeutic drugs for RA such as nonsteroidal anti-inflammation drugs (NSAID), steroids, glucocorticoids, immunosuppressants, and biological therapies are effective, they have significant side effects.7 For example, RA © XXXX American Chemical Society
patients are at risk of developing infections and certain malignancies after long-term treatment with biological agents.8,9 The treatments with methotrexate and low-dose prednisone also place patients at an increased risk of opportunistic infections.10 Therefore, novel effective while risk-free RA therapeutics are in urgent demand. In this regard, natural compounds with potent antioxidative and antiinflammatory activities represent invaluable resources for development as RA therapeutics.11−13 Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural polyphenol broadly existing in grapes, berries, peanuts, and mulberry and is particularly rich in Polygonum cuspidatum and the skin of red grapes.14 Resveratrol is noteworthy for its health-beneficial effects attributed by assorted mechanisms of Received: September 17, 2018 Revised: November 25, 2018 Accepted: November 25, 2018
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DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry action, including ROS scavenging,15,16 antioxidative,16,17 and anti-inflammatory activities,17,18 among others. To this end, a myriad of evidence has established the potential of resveratrol in the prevention or treatment of chronic inflammation-related disorders such as cardiovascular diseases, diabetes, obesity, and cancer.19−23 In view of that, we intended to address the question regarding the role of resveratrol in RA and the underlying mechanisms at both in vivo (bovine type-II collagen (BIIC)-induced rat arthritis model) and in vitro (interleukin (IL)-1β-stimulated rat synovial fibroblast-like cell model) experimental settings.24,25 Herein we presented evidence supporting the preventive effect of resveratrol on RA-induced injuries both in vivo and in vitro. Specifically, in rats with BIIC-induced arthritis, resveratrol markedly suppressed the pathological hallmarks of RA, including the synovial infiltration of inflammatory cells, oxidative stress, synovial hyperplasia, and neovascularization in synovial tissues. Further in vitro analysis in rat synovial fibroblast-like cells suggested that resveratrol alleviates RA, likely through inhibiting MAPK signaling pathways, HIF-1α expression, and cell proliferation along with the induction of apoptosis.
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swelling in all joints, and 4 = skin bursting/joint dysfunction or distortion.26 Clinical assessments were completed by two experimenters independently. The sum of arthritis index scores in the limbs was used to evaluate the severity of arthritis, with higher scores indicating more severe arthritis. Histological Assessment. Paraffin sections of joint tissues were stained with hematoxylin and eosin (H&E, Sigma-Aldrich) to examine the infiltration of inflammatory cells and pannus formation as well as to assess the severity of edema in the synovial tissue. The left hind limb specimens were fixed with 10% (v/v) neutral formalin for 24 h, embedded in paraffin, and sliced into 5 μm thick tissue sections. Then, H&E staining was carried out according to standard protocols. After staining, pathological changes in the joint tissues were observed at 200× magnification under a BH2 optical microscope (Olympus, Hino, Tokyo, Japan). The severity of RA was scored on a scale from 0 to 3, where 0 = normal, 1 = mild synovitis or slight cartilage erosion, 2 = moderate synovitis and cartilage erosion, and 3 = erosion of the cartilage or bone destruction.27 Determination of Serum MDA and SOD Levels. Blood samples from rats were naturally coagulated for 20 min after collection and then centrifuged at 1000 rpm for 10 min; the supernatant (serum) was collected and stored at −20 °C. Serum malonaldehyde (MDA) and superoxide dismutase (SOD) levels were determined using respective detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Absorbance was measured on an MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA). Cytokine Determination. Forty-two days after the initial immunization, all rats were euthanized by CO2 asphyxiation. Blood was immediately collected from five randomly selected rats from each group, and the serum was obtained after centrifugation at 1500 rpm for 10 min to determine cytokine levels thereafter. The levels of proinflammatory cytokines (IL-1β, MCP-1, IL-6, and TNF-α) were evaluated using enzyme-linked immunosorbent assay (ELISA) (BioSwamp, Wuhan, China), according to the manufacturer’s instructions. The optical density (OD) of each well was immediately read at 450 nm using a microtiter plate reader (Labsystems, Helsinki, Finland). Cell Culture.25 Rat synovial fibroblast-like cell line 364 (RSC-364, BioSwamp) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum and supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were maintained at 37 °C and 5% CO2. The medium was discarded when cells reached 70−80% confluency. Protein Extraction and Western Blotting. The lysates of RSC364 cells without drug treatment or treated with IL-1β (10 ng/mL) in the absence or presence of resveratrol (25 or 50 μmol/L) were harvested in ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride (BioSwamp). A bicinchoninic acid (BCA, BioSwamp) assay was employed for protein quantification. Protein extracts were subjected to 15% SDS−polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The PVDF membranes were blocked with 5% skim milk in Tris-buffered saline and then incubated at 4 °C overnight with appropriate primary antibodies. After three washes with Trisbuffered saline-Tween 20 (TBST), the PVDF membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (1:10 000) at 4 °C for 12 h. The antibody-reactive bands were visualized using enhanced chemiluminescence (ECL) and quantified with the ImageJ software (Media Cybernetics, Rockville, MD). Cell-Cycle Analysis. RSC-364 cells from each experimental group were harvested and fixed in ice-cold 99% aqueous ethanol at −20 °C until flow cytometry analysis. Prior to analysis on a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Brea, CA), the cells were washed with PBS and centrifuged at 800 rpm for 5 min. After centrifugation, the cells were suspended in 500 μL of a PI solution and incubated in the dark at 37 °C for 30 min. The content of PIlabeled DNA in the cells was quantified by flow cytometry. Cell Apoptosis Analysis. RSC-364 cells from each experimental group were harvested and washed three times with ice-cold PBS. The
MATERIALS AND METHODS
Animals. Thirty-two female Sprague−Dawley (SD) rats, aged 4−6 weeks, were obtained from Hubei Research Center of Laboratory Animals (Wuhan, China, certification no. SCXK (E) 2015−0018). The rats were given free access to food and water. All animal care procedures were followed, according to the Guidelines for the Care and Use of Laboratory Animals from the Ministry of Science and Technology of China. The animal experimental protocols used in this study were authorized by the Animal Care and Scientific Committee of Huanggang Normal University (Hubei, China). Chemicals. Resveratrol (98.5% purity) was acquired from Nanjing Spring & Autumn Biological Engineering (Nanjing, Jiangsu, China). Recombinant rat IL-1β was purchased from R&D Systems (Minneapolis, MN). Immunization-grade BIIC was obtained from Chondrex (Redmond, WA). Complete Freund’s Adjuvant (CFA) and Incomplete Freund’s Adjuvant (IFA) were purchased from SigmaAldrich (St. Louis, MO). Antibodies against HIF-1α, p-p38 MAPK, p38 MAPK, p-JNK, JNK, GAPDH, β-actin, and goat antirabbit IgG were all purchased from Abcam (Cambridge, London, England). Propidium iodide (PI), the Dead Cell Apoptosis Kit with Annexin VFITC and PI, enhanced chemiluminescence (ECL) reagents, and the BCA Protein Quantification Kit were obtained from Thermo Fisher Scientific (Waltham, MA). Establishment of BIIC-Induced Arthritis in SD Rats.24 After 7 days of adaption to laboratory conditions, all rats were randomly divided into four groups (8 rats/group), namely, drug-untreated control group (CON), BIIC-immunized group (BIIC), BIIC combined with resveratrol (200 mg/kg bw) treatment group (BIIC +R200), and BIIC combined with resveratrol (400 mg/kg bw) treatment group (BIIC+R400). Ten milligrams of BIIC and 5 mL of CFA were emulsified with a homogenizer, and 0.1 mL (100 mg collagen/mouse) of the emulsion was subcutaneously injected into the base of rat tails, except the CON rats. A booster injection of the emulsion of BIIC and IFA was performed 21 days after the initial immunization. Then, BIIC+R200 and BIIC+R400 rats received 200 and 400 mg/kg bw of resveratrol, respectively, by oral gavage feeding once daily, whereas CON and BIIC rats were instead fed orally with 1 mL of distilled water per gavage daily. Both distilled water and resveratrol were orally administered beginning 21 days after the initial immunization. The severity of arthritis in each involved paw from five randomly selected rats/group was scored using an established visual semiquantitative scoring system with scores ranging from 0 to 4 per paw, where 0 = the joint is normal, 1 = swelling in one joint (toe/ wrist/ankle/footpath), 2 = swelling in more than one joint, 3 = B
DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry cells were then resuspended and adjusted to a density of 1 × 106 cells/ mL with 1× Annexin-binding buffer. They were then fixed in 5 μL of Annexin V-FITC and 1 μL of 100 μg/mL PI. Thereafter, the cells were gently mixed and incubated in the dark at 4 °C for 30 min. Then, 400 μL of 1× Annexin-binding buffer was added. Finally, apoptosis of the cells in each group was observed by flow cytometry. Statistical Analysis. Statistical evaluation was performed by oneway analysis of variance (ANOVA) or a one-way Student’s t test and Duncan’s multiple range tests. Data are presented as the mean ± sd for the indicated number of independently performed experiments. A probability value of p < 0.05 or p < 0.01 was considered statistically significant.
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RESULTS Resveratrol Mitigated RA Severity in Rats with BIICInduced Arthritis. To assess the preventive effect of resveratrol on RA, we started by establishing an in vivo RA model using BIIC-induced arthritis in SD rats. It is clearly noted that BIIC stimulated the development of arthritis in a time-dependent manner, starting on the 31st day after initial immunization and reaching an arthritis score index of 8.37 on day 42 (Figure 1). With respect to BIIC-stimulated rats, on day
Figure 1. Effect of resveratrol on arthritis index scores in BIICimmunized SD rats. The average arthritis index score of each group is presented as the mean ± sd (n = 5 per group). The significance of differences among the four groups was analyzed by one-way ANOVA and Duncan’s multiple range test. Values that do not share a common superscript letter (a−c) are significantly different among groups when p < 0.01. CON, drug-untreated control group; BIIC, Bovine type-II collagen (BIIC)-treated group; BIIC+R200, BIIC combined with 200 mg/kg bw of resveratrol treatment group; BIIC+R400, BIIC combined with 400 mg/kg of resveratrol treatment group.
Figure 2. Effect of resveratrol on the articular cartilage tissue of BIICimmunized SD rats (200× magnification). (A) Representative H&E staining images (left panels) and paw morphology images (right panels) of each treatment group are shown. At the end of the experiment, left hind paw samples from five randomly selected rats in each group were harvested from the knee joint, and H&E staining was performed. Inflammatory cell infiltration and synovial destruction are marked by white arrows, whereas neovascularization is marked by black arrows. (B) Histological scores of each indicated treatment group. Different letters represent significant differences in the same indexes when p < 0.01.
31 there was no significant difference in arthritis severity between the BIIC alone group and the resveratrol-treated groups. Notably, the level of arthritis severity was markedly attenuated upon resveratrol treatment. In particular, on day 42 after initial BIIC immunization, the arthritis score index was dropped from 8.37 to 5.36 by 400 g/kg bw of resveratrol (p < 0.01) (Figure 1). Further histological analyses revealed that the arthritic joints of BIIC rats displayed tissue edema, severe inflammatory cell infiltration in the synovial tissue, and serious angiogenesis (vascular density) in the cartilage matrix, all of which were effectively alleviated after gavage feeding with 400 mg/kg bw of resveratrol (Figure 2A). To this end, we observed that the histological score of the BIIC rats reached to 1.73, which was reduced by nearly three-fold to 0.62 after treatment with 400 mg/kg bw of resveratrol (p < 0.01) (Figure 2B). Taken together, our results clearly demonstrated that resveratrol protected BIIC-stimulated rats from the formation of synovial tissue edema, the synovial infiltration of inflammatory cells, and the development of angiogenesis.
Resveratrol Lowered BIIC Immunization-Elicited Oxidative Stress and Inflammation in Vivo. Increased levels of oxidative stress and inflammation have been recognized as two fundamental elements for the pathogenesis of RA. In light of this, we first examined the effect of resveratrol on the status of oxidative stress revealed by the levels of MDA and SOD in the serum of BIIC-stimulated rats. Compared with those in control rats, BIIC treatment alone promoted a nearly 4.52-fold increase in MDA levels, whereas SOD levels were reduced to only 34.68% (p < 0.01) (Figure 3A). These results evidently demonstrated the induction of excessive oxidation in BIIC-stimulated RA rats. It is noteworthy that BIIC-elicited C
DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Effects of resveratrol on the serum levels of SOD and MDA (A) and proinflammatory cytokines (B) in BIIC-immunized SD rats. The average content of serum SOD, MDA, and cytokines in each treatment group is expressed as the mean ± sd (n = 5 per group). The significance of differences among groups was analyzed by one-way ANOVA and Duncan’s multiple range test. Different letters a−c represent significant differences in the same indexes when p < 0.01.
Figure 4. Effects of resveratrol on the protein expression of HIF-1α, p-p38 MAPK, p38 MAPK, p-JNK, and JNK in IL-1β-stimulated RSC364 cells. (A) Immunoblotting analysis for the expression levels of indicated proteins in each treatment group. (B) Quantitation analysis of immunoblotting signals shown in panel A. Protein expression levels were determined relative to GAPDH in three independent experiments. Data are presented as the mean ± sd (n = 3) and were statistically analyzed by one-way ANOVA and Duncan’s test. The relative expression level of each protein in drug-untreated cells was defined as 1. Different letters a−c indicate significant differences in the same indexes when p < 0.01.
alterations in serum MDA and SOD levels were reversed by the treatment of resveratrol dose-dependently, illustrating the inhibitory effect of resveratrol on ROS production (Figure 3A). To further address the effect of resveratrol on the inflammation status of BIIC-stimulated RA rats, the serum levels of proinflammatory cytokines were evaluated. We observed a dramatic increase in the levels of a panel of proinflammatory cytokines such as IL-1β, IL-6, MCP-1, and TNF-α following BIIC stimulation; nevertheless, all of these BIIC-induced proinflammatory cytokines were downregulated by resveratrol (p < 0.01) (Figure 3B), indicating that resveratrol counteracted the changes associated with BIICinduced inflammation. Collectively, these lines of evidence support the notion that resveratrol exerted both antioxidative and anti-inflammatory actions in the synovial tissue of RA rats. Resveratrol Abrogated IL-1β-Elicited HIF-1α Upregulation and MAPK Activation in RSC-364 Cells. We next investigated the mechanisms of action whereby resveratrol alleviates RA pathogenesis. Accumulating evidence has underscored the essential role of fibroblast-like synoviocytes in the pathological progression of RA, including synovial hyperplasia and inflammation, destruction of cartilage, and angiogenesis.28−30 In this regard, we tested the resveratrolmediated RA-protective effect in an in vitro RA model based on the rat fibroblast-like synoviocyte RSC-364 cell line stimulated by IL-1β.25 In RSC-364 cells, IL-1β elicited a marked increase in the protein level of HIF-1α, a master transcriptional regulator promoting angiogenesis, whereas the increase in HIF-1α was greatly reduced by cotreatment with resveratrol in a dose-dependent way (p < 0.01) (Figure 4A,B).
This finding indicated that resveratrol inhibited the upregulation of HIF-1α in IL-1β-stimulated RSC-364 cells, possibly leading to a blockade of HIF-1α-mediated VEGF production. Likewise, we observed that resveratrol blocked IL-1β-induced activation of both JNK and p38 MAPK, as evidenced by a decrease in the levels of activated JNK and p38 MAPK manifested by the dual phosphorylation at Thr183/Tyr185 of JNK (p-JNK) and at Thr180/Tyr182 of p38 MAPK (p-p38), respectively (Figure 4A,B). Previous studies have validated that ROS engages the JNK- and p38 MAPK signaling pathways to promote the expression of proinflammatory cytokines for triggering inflammation.6 Accordingly, it is likely that resveratrol suppressed IL-1β-induced ROS production to blunt the activation of JNK and p38 MAPK, thereby exerting its anti-inflammatory action. Resveratrol Inhibited Cell Proliferation and Induced Apoptosis in IL-1β-Stimulated RSC-364 Cells. Synovial hyperplasia is a prominent hallmark of RA.29 In view of that, we probed the effect of resveratrol on the proliferation and apoptosis of RSC-364 cells. As shown in Figure 5, IL-1β stimulation for 24 h caused 58.10 and 18.44% of the RSC-364 cell population to stay in the G0/G1 phase and the S phase of the cell cycle, respectively. However, after cotreatment with 50 μmol/L of resveratrol, the level of the G0/G1 population was enhanced to 76.38%, whereas that of the S phase was dropped D
DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 5. Effects of resveratrol on the cell-cycle progression of IL-1β-stimulated RSC-364 cells. Cells were treated without drugs or with IL-1β (10 ng/mL) in the absence or presence of 25 or 50 μmol/L of resveratrol for 24 h, followed by PI staining and flow cytometry analysis thereafter. (A) Representative histograms from flow cytometric analysis after PI staining in each treatment group. (B) Quantitation of the histograms shown in panel A. The data are presented as the mean (n = 3).
of RA include chronic synovial inflammation and pathological angiogenesis, causing progressive complex disability if untreated. Under the influences of chronic arthritic inflammation and synovial tissue hyperplasia, cartilage and bone will be progressively destroyed, leading to severe joint pain and functional impairment in RA patients. Oxidative stress is one of the most important pathological features of the synovial tissue.3 In arthritis patients, ROS are formed through the interaction between the cellular immune system and endogenous or exogenous antigens.31 In this study, we revealed elevated MDA contents with reduced SOD activity in the serum of BIIC-challenged rats, confirming increased oxidative stress in the in vivo RA model (Figure 3A). Moreover, BIIC stimulation provoked an inflammation status in vivo, as
to 9.63% (p < 0.01) (Figure 5). These data demonstrated that resveratrol delayed the proliferation of IL-1β-stimulated RSC364 cells by promoting the G0/G1 cell-cycle arrest. Additionally, we noticed that 5.21% of the RSC-364 cells underwent apoptosis after 24 h of stimulation with IL-1β, whereas the proportion of apoptotic cells was markedly increased to 19.48% after 50 μmol/L of resveratrol cotreatment (p < 0.01) (Figure 5). Overall, we concluded that resveratrol inhibited cell proliferation along with provoking apoptosis of IL-1βstimulated RSC-364 cells.
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DISCUSSION We herein presented evidence demonstrating the in vivo and in vitro preventive effect of resveratrol on RA. The characteristics E
DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry manifested by the enhanced synovial infiltration of inflammatory cells accompanied by a marked rise in the serum levels of proinflammatory cytokines (Figure 3B). Notably, the findings that resveratrol reversed BIIC-elicited alterations in MDA, SOD, and proinflammatory cytokines clearly demonstrated the strong antioxidative and anti-inflammatory activities of resveratrol to inhibit RA-associated ROS production and chronic inflammation in vivo, respectively, thereby alleviating RA severity (Figures 1 and 2). Angiogenesis is fundamental to RA progression due to its action to maintain chronic synovial inflammation and promote synovial hyperplasia.2 In the synovial tissue of RA patients, HIF-1α is continuously expressed at a high level and transferred to the nucleus for transactivating the expression of VEGF and other proteins.32,33 In this study, Western blotting results confirmed that the expression levels of HIF-1α in IL-1β-stimulated RSC-364 cells were considerably decreased after resveratrol treatment (Figure 4). Intriguingly, resveratrolinduced HIF-1α downregulation likely accounts for our observation that resveratrol inhibited angiogenesis in the synovial tissue of BIIC-immunized RA rats (Figure 2). How resveratrol downregulates HIF-1α in the context of RA is currently unknown. One possible mechanism is through its antioxidative action, given that ROS positively regulates HIF1α.34 An alternative mechanism is likely via its inhibitory effect on p38 MAPK (Figure 4), which promotes HIF-1α protein stability.35 It is also noteworthy that resveratrol was shown to inhibit HIF-1α accumulation and VEGF secretion in cobalt chloride (CoCl2)-induced human retinal pigment epithelial (hRPE) cells through the activity of SIRT1.36 Thus future studies are required to clarify the involvement of ROS, p38 MAPK, or SIRT1 in resveratrol-induced downregulation of HIF-1α. MAPKs, including ERK, JNK, and p38 MAPK, are pivotal to the pathogenesis of RA.6 In essence, RA-associated ROS elicits inflammation by activating MAPK signaling pathways, which, in turn, mediate the production of proinflammatory cytokines.4−6,37 p38 MAPK is especially prominent with respect to RA pathogenesis, because its activation contributes to almost all aspects of RA-related pathologies, including synovial inflammation, damage of cartilage and bone, and angiogenesis.38 On the contrary, JNK activity is closely associated with bone erosion in the early phase of RA.6 For those reasons, both p38 MAPK and JNK represent potential targets for RA therapeutics. In this regard, we demonstrated that resveratrol prevented the activation of both p38 MAPK in IL-1β-stimulated RSC-364 cells (Figure 4). Of note, the inhibition of p38 MAPK and JNK by resveratrol is likely a consequence of reduced ROS accumulation but also argues that functional blockade of p38 MAPK and JNK underlies the protective action of resveratrol on RA progression. Our study also uncovered that resveratrol concomitantly induced G0/G1 cell-cycle arrest (Figure 5) and apoptosis (Figure 6) in IL-1β-stimulated RSC-364 cells, a mechanism of action ostensibly responsible for the attenuation of RAassociated synovial hyperplasia. It is worth noting that JNK activity plays a key role in regulating the proliferation and survival of cells in the synovial tissues of RA. Specifically, JNK inhibition was associated with the blockade of cell proliferation in RA synovium after treatment with tocilizumab or golimumab, whereas inhibition of JNK-mediated suppression of FoxO1 led to the induction of apoptosis in IL-1β-stimulated fibroblast-like synoviocytes.39−41 Hence, it is plausible to
Figure 6. Proapoptotic effects of resveratrol on IL-1β-stimulated RSC-364 cells. Cells were treated without drugs or with IL-1β (10 ng/ mL) in the absence or presence of 25 or 50 μmol/L of resveratrol for 24 h, followed by Annexin V-FITC/PI dual staining and subsequent flow cytometry analysis. (A) Representative histograms of Annexin VFITC/PI dual staining. The top-right quadrant and the bottom-right quadrant represent Annexin V-FITC-stained cells (early-phase apoptotic cells) and PI- and FITC-dual-stained cells (late-phase apoptotic or necrotic cells), respectively. (B) Quantitation of the histograms shown in panel A. The values represent the mean ± sd of at least three independent experiments. Different letters a−d represent significant differences when p < 0.01.
reason that resveratrol blocks the activation of JNK to inhibit cell proliferation and to provoke apoptosis in RA-associated fibroblast-like synoviocytes. F
DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
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In conclusion, data presented here clearly demonstrated that resveratrol displays preventive effects for RA both in vivo and in vitro by reducing ROS accumulation, inflammation, and angiogenesis in the synovial tissue. Furthermore, the inhibition of MAPK signaling pathways likely accounts for the antiinflammatory, antiangiogenic, cytostatic, and proapoptotic actions of resveratrol. In this regard, resveratrol holds a great potential for clinical translation as a therapeutic agent for RA management.42
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AUTHOR INFORMATION
Corresponding Authors
*S.L.: E-mail:
[email protected]. Tel: 1-973-919-3702. Fax: 1-732-932-6776. *C.-C.C.:
[email protected]; chiachechang@ gmail.com. Tel: +886-4-22852022. Fax: +886-4-22853469. ORCID
Chi-Tang Ho: 0000-0001-8273-2085 Shiming Li: 0000-0002-6167-0660 Funding
This study received support from the High Level Scientific Research Cultivation Project of Huanggang Normal University (201615803) and the National Natural Science Foundation of China (31571832). Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.jafc.8b05047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
