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A study of Sirt1 regulation and the effect of resveratrol on synoviocyte invasion and associated joint destruction in rheumatoid arthritis

The aim of the current study was to investigate the role and mechanism of sirtuin 1 (Sirt1) in the regulation of synovial cell invasion and joint destruction in rheumatoid arthritis (RA). The Sirt1 protein and mRNA levels in fibroblast-like synoviocytes (FLS) isolated from RA synovial tissues were compared with normal tissues by western blot and reverse transcription-polymerase chain reaction. RA FLS were then treated with the Sirt1 agonist resveratrol (1, 3 and 10 µg/ml) for 48 h, and their invasiveness and expression of matrix metalloproteinase (MMP) 1 and MMP13 protein and mRNA were measured. Furthermore, a collagen-induced arthritis (CIA) rat model was established and the rats were divided into a model group, and low- and high-dose resveratrol (2.5 and 10 mg/kg/day) groups to receive an intraperitoneal injection of resveratrol for 42 consecutive days. The joint morphology, arthritis index (AI), and MMP1 and MMP13 expression in synovial tissues was monitored. The Sirt1 protein and mRNA levels in RA FLS were significantly lower compared with normal FLS (P<0.01). The resveratrol treatment significantly inhibited the invasive ability of RA FLS (P<0.01) and reduced MMP1 and MMP13 expression (P<0.01). The AI in low- and high-dose groups was significantly lower compared with the model group from day 28 (P<0.01). Resveratrol also reduced the swelling and damage and decreased MMP1 and MMP13 expression levels in CIA rats (P<0.01). The resveratrol-induced upregulation of Sirt1 in RA FLS may significantly inhibit the invasion of these cells and reduce the degree of joint damage, which may be mediated through the inhibition of MMP1 and MMP13 expression. The present results suggested a regulatory role for Sirt1 in RA pathogenesis, and demonstrated the beneficial effects of resveratrol, which may have potential as an alternative therapeutic strategy for the treatment of patients with RA.


Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects the peripheral joint symmetry, leading to erosive arthritis (1,2). RA is a widely prevalent disease with a high rate of morbidity and mortality. RA development gives rise to abnormal hyperplasia of synovial cells, forming synovial pannus with inflammatory cells and small blood vessels. Matrix metalloproteinases (MMPs) are secreted by the synovial pannus into synovial fluid and then transferred to the extracellular matrix, leading to degradation of articular bones and cartilages (3)(4)(5)(6).
Fibroblast-like synoviocytes (FLS) are the major cells in articular synovial tissues and important components of synovial lining tissues. FLS release multiple cytokines and have an important role in joint destruction. Recently, the invasion of FLS has been recognized as a novel target for RA therapy (3)(4)(5)(6). Sirtuin 1 (Sirt1) is one of the most well-studied members of the sirtuin protein family. As a nicotinamide adenine dinucleotide + -dependent histone deacetylase present in all mammalian somatic cells, the Sirt1 protein is involved in several diseases, including neurodegenerative diseases, diabetes, cancer and inflammation. It has been reported that Sirt1 exhibits abnormal expression in RA synovial tissues and has an important role in the development of the disease (7)(8)(9). Furthermore, the therapeutic effects of Sirt1 agonists have been observed in mice with collagen-induced arthritis (CIA) (10)(11)(12). Sirt1 is also associated with the invasiveness of a variety of tumor cells (13,14). We therefore hypothesize that Sirt1 may be closely associated with RA FLS invasion and Sirt1 expression level may directly affect the invasiveness of these cells. In this study, the expression level of Sirt1 protein and mRNA in RA FLS was examined by western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Resveratrol has been known as an Sirt1 agonist due to its ability to enhance the deacetylase activity of Sirt1 (15). The RA FLS were then treated with resveratrol for 48 h, and their invasiveness and MMPs expression levels were measured. Further, a CIA rat model was established to evaluate effect of Sirt1 activation by resveratrol on the arthritis index (AI), joint morphology and MMP expression levels in these rats.
Synovial tissues. Rheumatoid synovial tissues were collected from 20 patients with rheumatoid disease that underwent knee replacement surgery in the Department of Orthopedics, The Second Affiliated Hospital of Nanchang University (Nanchang, China) between December 2013 and December 2015. All patients met the classification standard of American Rheumatology Society (16). Normal synovial tissues were obtained from non-rheumatoid patients that had undergone orthopedic amputations due to accidental injury. The study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University. All patients were required to provide signed informed consent.
In vitro cell experiments FLS isolation and culture. The collected synovial tissues were washed three times with PBS under sterile conditions. After removing fat with a blade, the tissues were cut into small pieces with surgical scissors and digested with 0.25% trypsin and 3% collagenase for 30 min-3 h to obtain single cell suspensions. The cells were re-suspended in DMEM medium (10% FBS, 100 U/ml penicillin-streptomycin) and incubated at 37˚C in an incubator with 5% CO 2 . Cell fusion occurred after 5 or 6 days, and cells at passage 2-3 were used for subsequent experiments.
Cell invasion assay. The RA FLS in the logarithmic growth phase (1x10 4 cells) were seeded on 6-well plates. After 24 h, they were treated with 1, 3, and 10 µg/ml resveratrol, and untreated RA FLS were used as the control. After 48 h, the cells were trypsinized and added to the upper chamber of a Transwell device in serum-free DMEM, and the lower chamber was filled with 5% DMEM containing 10% FBS. The device was incubated for another 24 h. The lower chamber was removed, washed, fixed with 0.4% paraformaldehyde at room temperature for 5 min, and stained with crystal violet. Cell invasion was measured by counting the number of invaded cells under an inverted optical microscope. Mean values were obtained from five randomly selected visual fields for each well.
Cell viability assay. RA FLS in the logarithmic growth phase were seeded in 96-well plates at a density of 6x10 3 cells/well. After 24 h, they were treated with 1, 3, and 10 µg/ml resveratrol, and untreated RA FLS were used as the control group. After 48 h, 20 µl 5 mg/ml MTT was added, followed by an addition of 150 µl of DMSO after 4 h. The optical density value representing the cell viability was then measured with a microplate reader (Bio-Rad Laboratories, Inc.) at 560 nm.

In vivo animal experiments
Modeling and grouping. A total of 50 4 Sprague-Dawley (SD) rats (age, 4 weeks; weight, 200±20 g) were purchased from Slack Animal Experiments, LLC [Shanghai, China; certificate: SCXK (Shanghai) 2012-0002]. The rats were housed at 23±2˚C under a 12-h light/dark cycle with free access to food and water. The CIA rat model was established as previously described (17). Briefly, the rats were given a subcutaneous injection of 100 µl 2 g/l type II collagen emulsion in the right-back toe pad on day 1 and a booster injection of the same dose on day 7. The normal group received an equal volume of saline at the same time points. The CIA rats were randomly divided into model group, low-dose resveratrol group, and high-dose resveratrol group (n=10 rats/group). The low-dose and high-dose groups were intraperitoneally injected with 2.5 and 10 mg/kg resveratrol per day for 42 consecutive days, respectively. The normal and model groups were injected with an equal amount of saline. The animal study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.
AI assessment. The AI of each rat was assessed as previously described (18). Briefly, the hind limb and joint were observed at intervals of 7 days after the first day of the model. The joints were evaluated with a scale of 0-4, for which 0 indicates normal joint, 1 for mild joint swelling, 2 for moderate swelling, 3 for severe joint swelling incapable of bending and walking, and 4 for severe swelling incapable of weight-loading. Joint X-ray assessment. All rats were subjected to joint X-ray examination at day 42 (19). Each rat was anesthetized by an intraperitoneal injection of 70 mg/kg of 0.6% sodium pentobarbital, and the right-back toe was examined for joint destruction using a dental X-ray machine (with a setup of 1 sec, 60 KV, 3 mA).
Collection of synovial tissues. At day 42, rats were sacrificed. The synovial tissues were collected, homogenized with a homogenizer and subjected to subsequent experiments.
Western blot analysis. The expression levels of MMP1, MMP13 and Sirt1 in RA FLS and synovial tissues in CIA rats were determined by western blot analyses. Briefly, total protein was extracted using protein extraction kits and quantified using a BCA kit according to the manufacturer's instruction. Equal amounts of total protein (10 µg) were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membrane was blocked in TBS buffer containing 5% skim milk and 0.1% Tween-20 at room temperature for 1 h, and incubated with the following primary antibodies overnight at 4˚C: Anti-MMP-1, anti-MMP-13, anti-sirt1 (1:100) and anti-GAPDH (1:200). The membrane was washed three times with TBST for 5 min each, and incubated with HRP-labeled goat anti-rabbit or goat anti-mouse IgG (1:500) at room temperature for 1 h. The membranes were washed three times with TBST and subjected to enhanced chemiluminescent detection. The intensity of bands was detected by a Molecular Imager ® ChemiDocTM XRS System (Bio-Rad Laboratories, Inc.). The gray value of bands was analyzed by Image Lab 2.0 software (Bio-Rad Laboratories, Inc.). The relative expression level of target protein was calculated as the ratio of gray value of target protein to that of the internal control (GAPDH). The experiments were performed in triplicate.
RT-PCR analysis. The mRNA levels of MMP1, MMP13 and Sirt1 in synovial tissue and synovial cells were detected by RT-PCR analyses. Total RNA in RA FLS or synovial tissue was extracted from with TRIzol reagent (Invitrogen, Shanghai, China) according to the manufacturer's instructions. RNA concentration was determined using a spectrometer under a wavelength of 260 nm. The total RNAs were reverse transcribed into cDNA using the HiFi Script cDNA kit (CWBio, Beijing, China) according to the manufacture's protocol. The temperature protocol that was used was as follows: at 50˚C for 1 h and at 80˚C for 5 min. qPCR amplification was performed on cDNA using Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, China). The primer sequences were as follows: Sirt1, forward 5'-TGG ACT CCA CGA CGT ACT, reverse 5'-TCT CCT GGG AGG CAT AGA CC; MMP1, forward 5'-ACT CTG GAG TAA TGT CAC ACC T, reverse 5'-GTT GGT CCA CCT TTC ATC TTC A; MMP13, forward 5'-ATG GAT GTC TTC AAG AAG GG, reverse 5'-CTA GTC TCC CCC ACT CTG GG; GAPDH, forward 5'-AGC CAC ATC GCT CAG ACA, reverse 5'-TCT CCT GGG AGG CAT AGA CC. The PCR program was as follows: 95˚C for 4 min, followed by 40 cycles of 95˚C for 40 sec, 58˚C for 30 sec, and 72˚C for 30 sec. The PCR products (5 µl) were examined on 2% agarose gel, and the nucleotide bands were detected with a gel imaging system (Bio-Rad Laboratories, Inc.). The DNA marker (D0107) was purchased from Beyotime Institute of Biotechnology.
Statistical analysis. Three replicates were performed for each test and all data were expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences among groups were compared by one-way analysis of variance, followed by least significant difference test. P<0.05 was considered to indicate a statistically significant difference.
Resveratrol upregulates Sirt1 expression. As presented in Fig. 2, treatment of RA FLS with resveratrol (1, 3 and 10 µg/ml) significantly increased the expression level of Sirt1 mRNA and protein compared with the control group (P<0.01). The highest Sirt1 expression was observed in the 10 µg/ml resveratrol group, followed successively by the 3 and 1 µg/ml groups, suggesting a dose-dependent stimulatory effect of resveratrol on Sirt1 expression. Effect of Sirt1 upregulation on RA FLS invasiveness. RA FLS cell invasiveness in different groups was compared by a cell invasion assay. As demonstrated in Fig. 3, the number of invading cells in the treated groups (90.3±10.0, 71.2±7.7, and 63.3±6.4 for the treatment of 1, 3, and 10 µg/ml resveratrol groups, respectively) was significantly lower compared with the sham control group (183.4±10.2, P<0.01). A significant difference in the number of invasive cells was detected among the three resveratrol groups (P<0.01), suggesting that the induction of Sirt1 by resveratrol may significantly inhibit the invasion ability of RA FLS cells in a dose-dependent manner. Furthermore, RA FLS cell viability was detected by MTT assay. There was no significant difference in the cell viability among different groups, indicating that resveratrol did not exert a significant impact on the viability of RA FLS cells (P>0.05; Fig. 4).

Effect of the Sirt1 upregulation on the expression level of MMPs in RA FLS.
The expression level of MMP1 and MMP13 protein was detected by western blot analyses. As demonstrated in Fig. 5, the MMP1 and MMP13 protein levels in RA FLS treated with resveratrol (1, 3 or 10 µg/ml) was significantly reduced compared to the control group (P<0.01). Consistently, the resveratrol treatment (1, 3, or 10 µg/ml) also significantly decreased the MMP1 and MMP13 mRNA level in RA FLS (P<0.01; Fig. 6). It was also shown that the MMP1  and MMP13 protein and mRNA level in 1 µg/ml resveratrol group was the highest, followed successively by 3 and 10 µg/ml groups, indicating that resveratrol dose-dependently inhibited the MMP1 and MMP13 protein and mRNA expression, suggesting that Sirt1 upregulation may reduce the expression of these MMPs.

Effect of resveratrol on AI in CIA rats.
Of the 50 rats in the study, 40 rats exhibited a marked loss of appetite, activity and hair gloss compared to the normal healthy group, thus producing an 80% success rate in establishing the CIA model. Furthermore, 30 CIA model rats were randomly selected and grouped into model group, low-dose resveratrol group and high-dose resveratrol group. Sirt1 expression in the synovial tissue of CIA rats was analyzed by western blotting, as shown in Fig. 7: Resveratrol at low and high doses significantly upregulated Sirt1 expression in the synovial tissue of CIA rats (P<0.01). Furthermore, Sirt1 expression in the high-dose group was significantly higher compared with in the low-dose group (P<0.01). The rat AI was monitored throughout the treatment. As presented in Fig. 8, the AI in the model group began to increase after the first day of modeling, reached the highest level at day 21, and subsequently plateaued (P<0.01, vs. normal group). The AI in both the low-and high-dose groups began to decrease compared with the model group after day 21, and reached the lowest levels at day 42. The AI in the low-and high-dose groups were significantly reduced compared with the model group at day 28, 35 and 42 (P<0.01), and also the AI in high-dose group was significant lower compared with the low-dose group at these time points (P<0.01).
Effect of the resveratrol on joint morphology in CIA rats. The right-back toe of each rat was examined for joint destruction by X-ray at day 42 of the model. In the normal control group there was normal bone tissue, clearly visible space between the toes, a sharp bone edge and uniform bone mineral density. By contrast, swollen bone tissue, narrowed joint space, blurry bone edge and uneven bone density were observed in the model group. Markedly reduced bone tissue swelling and improved joint space were observed in the high and low dose resveratrol groups compared with the model group, suggesting that Sirt1 upregulation by resveratrol may reduce joint destruction in CIA rats.
Effect of resveratrol on MMP expression levels in the synovial tissue of CIA rats. The expression level of MMP1 and MMP13 protein and mRNA in the synovial tissue of CIA rats was detected by western blot analyses and RT-PCR. As presented in Fig. 9, the MMP1 and MMP13 protein expression in the model group was significantly higher than in the normal group (P<0.01). Both the high and low dose of resveratrol significantly reduced the level of MMP proteins compared with the model group (P<0.01). Consistently, the resveratrol treatment at either low or high dose significantly reduced the expression level of MMP1 and MMP13 mRNAs in the synovial tissue in the CIA rats as compared to the model group (P<0.01; Fig. 10). A dose-dependent inhibitory effect of resveratrol on the MMP1/13 protein and mRNA expression was also observed, indicating that the resveratrol-induced Sirt1 upregulation may mediate the reduced expression of these MMPs. The expression of MMP1 and MMP13 mRNA and protein in high-dose group was significantly reduced compared with low-dose group (P<0.01).

Discussion
The silence information regulator 2 (Sir2) gene family of deacetylases in yeast chromatin broadly participate in several biological processes, including cell survival, apoptosis and senescence. As a homolog of Sir2, Sirt1 also has deacetylase activity, and interacts with a variety of histone and non-histone proteins to maintain a low level of acetylation in normal cells, and normal expression level of non-histone proteins. Sirt1 is closely associated with a variety of physiological and pathological processes, including cell apoptosis, cell cycle, cell energy metabolism, lipid accumulation and cellular senescence (20).
Although several studies have detected abnormal expression of Sirt1 in RA patients, the role of Sirt1 expression in RA has remained controversial. For instance, Wendling et al (8) reported that the Sirt1 protein level in peripheral blood mononuclear cells from RA patients is lower that the levels in healthy people. Wang et al (21) reported that induction of the Sirt1 level via inhibition of nuclear factor-κB (NF-κB) p65 protein expression improves arthritis in CIA rats. By contrast, Niederer et al (9) reported that Sirt1 was overexpressed in RA synovial tissue compared with OA. Silencing of the sirt1 gene promotes apoptosis of RA FLS, whereas overexpression of this sirt1 can represses cell apoptosis. In the current study, the Sirt1 mRNA and protein level in RA FLS was examined by western blot and RT-PCR analyses. The results demonstrated a significantly lower level of Sirt1 expression in RA FLS than in normal control FLS, which is consistent with the study by Wendling et al (8). The current study also revealed that resveratrol dose-dependently increased the level of Sirt1 protein and mRNA in RA FLS.
As a main component of synovial cells, FLS are located in the synovial lining layer and possess 'tumor cell-like' invasive biological behavior. A previous study suggested that RA joints secrete a large amount of inflammatory cytokines and MMPs into the extracellular matrix under low oxygen conditions, leading to the destruction of articular cartilage, bone and joints (22). Lefevre et al (23) reported that RA FLS migrate within intra-articular regions and also across the vascular endothelium, causing degradation of articular cartilage. Videographic examination demonstrated that joints in RA patients with more aggressive FLS are more vulnerable to damage and have higher sharp scores compared with patients with mild or medium FLS (24). Therefore, inhibition of the migration and invasion capacity of RA FLS is expected to become a novel target for the treatment of RA. In this study, resveratrol dose-dependently inhibited the invasion of RA FLS although it did not affect the viability of these cells, indicating that Sirt1 upregulation by resveratrol may markedly reduce the invasive capacity of RA FLS. Previous studies have suggested that resveratrol may exert anti-inflammatory and therapeutic effects in RA through the modulation of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt pathway (25) and acetylation of activator protein-1 and nuclear factor (NF)-κB (26).
MMPs, a class of zinc-dependent proteolytic enzymes that are secreted by RA FLS, degrade extracellular matrix, bone   and cartilage, and thus, have an important role in the joint destruction that occurs during RA. High expression levels of MMPs, including MMP1, MMP2 and MMP9, are detected in RA synovial fluid and tissue (27). Aggressive expression of MMP3 and MMP13 was also detected in RA-FLS isolated from patients with RA (28). Furthermore, the migration and invasion ability of RA FLS are significantly inhibited through the downregulation of MMP1 and MMP13 expression (29,30). The current study demonstrated that MMP1 and MMP13 expression in RA FLS was reduced after the induction of Sirt1 by resveratrol at different doses, suggesting that Sirt1 may influence the invasiveness of RA FLS through the regulation of MMP1/13 expression. Nevertheless, further knockdown and overexpression experiments are required to confirm this speculation. Additionally, Sirt1 has been previously demonstrated to inactivate the NF-κB signaling pathway through deacetylation of the pathway subunit RelA/p65 to affect the expression of downstream target proteins, such as the inflammatory cytokines TNF-α, IL-1β and MMPs (31). Therefore, the association between Sirt1 and the NF-κB signaling pathway in RA may be an important future research focus. A CIA rat model was established to evaluate the effect of resveratrol-induced Sirt1 upregulation in the treatment of RA. Similar to that in a previous report (17), a modeling success rate of 80% was achieved. The AI value in the model group was gradually increased and peaked at day 21, when the most severe joint swelling was also observed during X-ray examination. The rats in the treatment groups received low-or high-dose resveratrol for 42 days, and subsequently the AI, joint morphology, and MMP expression levels in synovial tissue were monitored. The AI in both the low-and high-dose groups began to decrease after day 21 of the model, and reached the lowest level at day 42. At day 42 of the model, X-ray examination of joint morphology demonstrated that resveratrol treatment at both doses reduced the joint swelling and bone destruction, and improved joint spaces and the bone edge. These results confirmed that upregulation of Sirt1 by resveratrol may decrease the AI in CIA rats, restore the joint morphology and reduce the joint destruction. Consistently, Kim et al (10) have also reported a therapeutic effect of resveratrol in CIA rats. Furthermore, the MMP1 and MMP13 expression in synovial tissue were substantially reduced by resveratrol treatment, indicating that the therapeutic effect of resveratrol may be associated with the downregulation of MMP protein.
In summary, the in vitro experiments in the present study revealed that Sirt1 expression was lower in RA FLS compared with normal FLS, and an upregulation of Sirt1 expression in RA  FLS was achieved by treatment with resveratrol. Resveratrol treatment also significantly inhibited the invasiveness of RA FLS, potentially by reducing the expression of MMP1 and MMP13. Further animal studies confirmed that the induction of Sirt1 by resveratrol significantly decreased the AI value, restored joint morphology, reduced joint damage and lowered the MMP1/MMP13 expression in CIA rats, suggesting that Sirt1 may regulate synoviocyte invasion and the associated joint destruction by inhibiting the expression of MMP proteins. The present results suggested a regulatory role for Sirt1 in RA pathogenesis, and demonstrated the beneficial effects of resveratrol, which may have potential as an alternative therapeutic strategy for the treatment of patients with RA.