The Effect of Resveratrol in Sirt1/CST Pathway to Inhibit TNF-α Induced Inflammatory Response in Rat Primary Fibroblast-Like Synoviocytes

Abstract

Rheumatoid arthritis has a significant impact on the life quality, but current pharmacological therapies have limitations. As a result, there is growing interest in exploring the potential of natural plant components to intervene in the development of rheumatoid arthritis. Resveratrol, a natural polyphenol and one of the main active components of the Chinese herbal medicine Polygonum cuspidatum, has emerged as a promising candidate for this purpose. In the present study, we investigated the role and mechanism of resveratrol in inhibiting inflammatory response in rat primary fibroblast-like synoviocytes. Tumor necrosis factor (TNF)-α was used to establish a model of inflammation, the Sirtuin1 selective inhibitor Selisistat (EX527) was used to inhibit Sirtuin1 activity, and small interfering RNA was used to silence cortistatin expression. The results showed that pre-treatment with resveratrol could time- and dose-dependently inhibit TNF-α induced cellular interleukin (IL)-1β and IL-6 secretion, and upregulate Sirtuin1 and cortistatin mRNA and protein expression in the range of 48 h, 100 µM. Selisistat (EX527) could attenuate resveratrol inhibited inflammatory response and upregulated cortistatin expression. Silencing cortistatin expression attenuated the effect of resveratrol on inhibiting inflammatory response, but did not affect its effect on upregulating Sirtuin1 expression. In conclusion, resveratrol effectively inhibited the TNF-α induced inflammatory response in fibroblast-like synoviocytes by a mechanism involving the Sirtuin1/cortistatin pathway.

INTRODUCTION

Rheumatoid arthritis (RA) is an autoimmune disease characterized by symmetrical joint damage, which leads to progressive joint destruction and even disability and seriously affects individuals’ QOL.¹⁾ It is now generally accepted that chronic inflammation of the synovial membrane is the centre of its multiple pathological changes.^2,3) Inflammation induces changes such as synovial proliferation, formation of vascular opacities, and destruction of cartilage tissue, ultimately leading to joint deformity and functional impairment. Cortistatin (CST), a member of the growth inhibitor family, has recently been found to inhibit tumor necrosis factor (TNF)-α production and competitively bind to TNF-α receptors, exerting anti-inflammatory effects and reducing collagen-induced joint swelling and cartilage destruction in mice.^4,5) However, the biological characteristics of cortistatin, peptides inconvenient for oral for instance, limit its application. Obtaining active ingredients from natural plants that modulate endogenous CST, then inhibit synovial inflammatory response is promising in the prevention and treatment of RA.

Resveratrol (Res), a natural polyphenol, is widely found in a variety of plants such as grape seeds and Polygonum cuspidatum.⁶⁾ Cardiovascular protector, is the first recognition most people have of resveratrol, originated from the French paradox phenomenon.⁷⁾ In China, Polygonum cuspidatum can clear dampness and heat, dispel wind and promote the circulation of the ligaments, which are used for rheumatic paralysis and pain was first recorded in masterpieces of Chinese Traditional Medicine Mingyi Bielu.⁸⁾ Now, Polygonum cuspidatum frequently used as the Principal Drug or Ministerial Drug in many experienced prescriptions for the treatment of RA in Chinese Traditional Medicine.^9–11) As one of the main active ingredients in Polygonum cuspidatum, resveratrol can also inhibit collagen-induced inflammatory response of joints in rat RA model in previous studies, which the mechanism involves upregulation of endogenous CST expression.¹²⁾ Several studies have demonstrated that resveratrol is a natural agonist of silencing information regulator 1 (Sirtuin1, Sirt1),^13,14) which plays an important role in the development of RA by inhibiting the activation and proliferation of macrophages and T cells, reducing cytokine secretion and thereby reducing the inflammatory response in joints through various mechanisms.^15,16) Although there are many clues suggesting a mechanism, whether the role of resveratrol in suppressing the inflammatory response of RA and upregulating CST expression is related to Sirt1 has not been reported. In this study, we investigated whether resveratrol exerts its anti-inflammatory effect through its mediated Sirt1 agonistic effect in primary cultured rat fibroblast-like synovial cells, and explore whether this effect involves endogenous CST regulation, in order to provide new directions for the clinical treatment of RA.

MATERIALS AND METHODS

Cell Extraction, Culture, and Identification

SPF Sprague-Dawley (SD) rats, body weight 280–320 g, were purchased from Hunan SJA Laboratory Animal Co., Ltd. (SCXK 2021-0002). The experiment was reviewed by the Animal Ethics Committee of Hunan University of Chinese Medicine and conducted in accordance with the Regulations on the Administration of Laboratory Animals.

Rats were anaesthetized, bled and executed, then the synovial tissue of knee joint was isolated. Rat fibroblast-like synoviocytes were extracted according to the method we reported previously,¹²⁾ and cultured in Dulbecco’s modified Eagle’s medium (DMEM) high sugar medium (HyClone, U.S.A.) containing 10% fetal bovine serum (Gibco, U.S.A.) and 1% penicillin–streptomycin solution (Invitrogen, U.S.A.) at 5% CO₂ and 37 °C. When the primary cells covered more than 80% of the bottom of the bottle, trypsin was digested and passaged. After cells were passed to 3 generations, they were identified as fibroblast-like synovial cells by immunocytochemical staining with Vimentin and CD68. Mouse anti-rat Vimentin monoclonal antibody and rabbit anti-rat CD68 polyclonal antibody are all from Boster (Wuhan, China). Phosphate buffered saline (PBS) was used instead of primary antibody as a negative control to exclude secondary antibody non-specific staining.

Small Interfering RNA (siRNA) Transfection for Silencing CST

CST siRNA and negative siRNA were designed and constructed by RIBBIO Co., Ltd. (Guangzhou, China). To ensure success, RIBBIO offer three pairs of CST siRNA. Sequences 1: forward 5′-CGGCCUUCUGACUUUCCUUdTdT-3′ and reverse 3′-dTdTGCCGGAAGACUGAAAGGAA-5′. Sequences 2: forward 5′-GUCUAAGCGGCAGGAAAGAdTdT-3′ and reverse 3′-dTdTCAGAUUCGCCGUCCUUUCU-5′. Sequences 3: forward 5′-CCUUCUCCUCGUGCAAGUAdTdT-3′ and reverse 3′-dTdTGGAAGAGGAGCACGUUCAU-5′.

Configure 0.1 mL transfection system according to transfection reagent (Fermentas, U.S.A.) instructions. One point eight milliliters complete medium and 0.2 mL transfection system were added to each well of the 6-well plates. The same ratio of medium was used for other cell culture plates, ensure the final concentration of siRNA was 100 µM. After 6 h, the cell culture medium was removed and replaced with fresh complete medium to continue the culture. After 24 h of transfection, cells or cell supernatants were collected for the next experiment. The negative siRNA transfection group was used as the negative control. Real-time PCR and immunofluorescence were used to detect CST mRNA and protein expression in cells. Transfection efficiency >70% compared with negative siRNA group was considered successful silence.

Grouping and Intervention

TNF-α is a predominant inflammatory mediator in RA, and is directly involved in the synovial inflammatory response. To investigate the role and mechanism of resveratrol in inhibiting inflammatory response, TNF-α was used to develop a model of inflammation. With this model, we designed three experiments. Each experiment was repeated three times independently.

The experiment 1 designed to observe the protective effect of resveratrol on inflammatory response induced by TNF-α. Exp 1 was divided into four groups: Control group, Model group, Res group, and Vehicle group. Cells in the Control group were cultured without any intervention. The Model group cells were incubated with 20 ng/mL recombinant rat TNF-α protein (Peprotech, U.S.A.) for 24 h to develop a model of inflammation. The cells in Res group were pretreated with 10, 30, 100 µM resveratrol (Kanglong, Changsha, China, purity ≥ 99%) for 24 h or 100 µM resveratrol for 12, 24, 48 h, then stimulated with 20 ng/mL TNF-α for 24 h to observe the protective effect of resveratrol. In the Vehicle group, an equal volume of Res solvent (ethanol) replaced Res solution for 24 h culture, then stimulated with 20 ng/mL TNF-α for 24 h to exclude the interference of the resveratrol’s solvent.

The experiment 2 was to address whether Sirt1 was involved in the effect of resveratrol by using the Sirt1 specific inhibitor of Selisistat (EX527). Exp 2 was divided into five groups. They are Control group, EX527 group, Model group, Res group, and Res + EX527 group. The Control group regular cultured without any intervention. The EX527 group was given 10 µM EX527 (MedChemExpress, U.S.A.) to incubate the cells for 24 h. The treatment of the Model group and Res group in experiment 2 are identical to experiment 1. The Res + EX527 group was pre-treated with 100 µM Res + 10 µM EX527 for 24h, then challenged with 20 ng/mL TNF-α for 24 h to determine the involvement of Sirt1 in the anti-inflammatory effect of resveratrol.

The aim of the experiments 3 was to observe if CST siRNA can decrease or abolise the anti-inflammatory effect of resveratrol. Exp 3 divided into six groups of Control group, CST siRNA group, Model group, Res group, Res + CST siRNA group, and Res + negative siRNA group. The CST siRNA group was transfected by CST siRNA for 24 h. The Res + CST siRNA group was transfected by CST siRNA for 24 h, 100 µM Res incubation for 24 h, then stimulated with 20 ng/mL TNF-α for 24 h to observe the role of CST in the anti-inflammatory effect of resveratrol. The Res + negative siRNA group was transfected by negative siRNA for 24 h, 100 µM Res incubation for 24 h, then stimulated with 20 ng/mL TNF-α for 24 h to ensure that all the results in the Res + CST siRNA group came from CST silencing rather than the transfection process. The treatment in the rest three groups in Exp 3 are identical as in Exp 1.

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of interleukin (IL)-1β and IL-6 in cell supernatants were measured according to the instructions of the ELISA kits (BOSTER, Wuhan, China), and detected by DLX-880 enzyme immunoassay instrument (BIO-TEK, U.S.A.).

Fluorescence Quantitative Real-Time PCR

Trizol (TaKaRa, Dalian, China) extracted the total RNA in cells, synthesized cDNA with reverse transcription kit (TaKaRa) by PCR instrument (Biometra, Germany), then diluted 25 times. Gene fragments were amplified by 7300 fluorescence quantitative real-time PCR (real-time qPCR) instrument (Applied Biosystem, U.S.A.) with 2 µL of diluted cDNA as template and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference, while a negative control without cDNA was set. Real-time PCR parameters were set based on the previous work, and the relative values of IL-1β, IL-6, Sirt1 and CST mRNA expression in the cells were detected. The following primers were used in this study: forward primer 5′-TGCTGATGTACCAGTTGGGG-3′ and reverse primer 5′-CTCCATGAGCTTTGTACAAG-3′ for IL-1β, forward primer 5′-GCCCTTCAGGAACAGCTATG-3′ and reverse primer 5′-CAGAATTGCCATTGCACAAC-3′ for IL-6, forward primer 5′-TCCTTTCAGAACCACCAA-3′ and reverse primer 5′-GGCGAGCATAAATACCAT-3′ for Sirt1, forward primer 5′-CCGGCCTTCTGACTTTCCTT-3′ and reverse primer 5′-TGCTGGAGGGGTGGTCTTT-3′ for CST, forward primer 5′-CCCCCAATGTATCCGTTGTG-3′ and reverse primer 5′-TAGCC CAGGATGCCCTTTAGT-3′ for GAPDH.

Western Blot

After adding cell lysis solution and lysing the cells by ultrasound, the supernatant was extracted by centrifugation. The final concentration of sample protein was detected and quantified to 4 µg according to the instructions of bicinchoninic acid (BCA) kit (Beyotime, Beijing, China), and denatured at 99 °C for 10 min. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel was prepared according to the instructions of gel-making kit, and polyvinylidene difluoride (PVDF) was transferred to the membrane after electrophoresis (Liuyi, Beijing, China). Closed membrane with 5% skimmed milk powder for 1 h at room temperature. After TBST rinsing, specific primary antibodies, rabbit anti-rat Sirt1 polyclonal antibody (Santa Cruz, U.S.A.) (1 : 1000) and mouse anti-rat GAPDH monoclonal antibody (Millipore, U.S.A.) (1 : 5000) were added and incubated overnight at 4 °C. After rinsing with TBST, the corresponding secondary antibodies (Beyotime) (1 : 5000) were added and incubated for 1 h at room temperature. After development (Bio-RAD, U.S.A.), Image J software analyzed the samples for grayscale, and GAPDH was used as an internal reference to detect Sirt1 protein expression in the cells.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 15 min at 37 °C, rinsed with PBS and incubated with 0.1% TritonX-100 for 10 min at room temperature, rinsed with PBS and then closed with 5% BSA blocking solution for 45 min at 37 °C. Goat anti-rat CST polyclonal antibody (Santa Cruz) (1 : 50) was added and incubated overnight at 4 °C in a wet box. After rinsing with PBS, fluorescent secondary antibody (Beyotime) (1 : 500) was added and incubated for 1 h at 37 °C protected from light. After PBS rinsing, CST protein expression in the cells was observed under fluorescence microscope (Nikon, U.S.A.). PBS was used instead of primary antibody as a negative control to exclude secondary antibody non-specific staining.

Data Statistics and Analysis

Data were expressed as mean ± standard deviation (S.D.). SPSS 26.0 software was used for statistics, and GraphPad Prism 9 software was used for graphs. Differences between groups were analyzed by One-Way ANOVA, p < 0.05 was considered statistically significance.

RESULTS

Observation and Identification of Rat Fibroblast-Like Synoviocytes

Primary fibroblast-like synoviocytes were isolated and cultured from rat synovial tissues, and spindle-shaped or polygonal cells were observed. After 3 to 5 generations, cells appeared mainly spindle-shaped with uniform translucent cytoplasm. As shown in Fig. 1, the cells were positive for Vimentin expression and negative for CD68 expression, exhibiting typical characteristics of fibroblast-like synoviocytes. Negative controls were used to exclude non-specific staining from secondary antibodies.

Fig. 1. Immunohistochemical Identification of Rat Fibroblast-Like Synoviocytes

Resveratrol Inhibits TNF-α Induced Inflammatory Response

As shown in Figs. 2A–C, the levels of inflammatory factors IL-1β and IL-6 secreted by cells in the Model group were substantially increased compared with the Control group (p < 0.01). Different concentrations of Res pre-intervention could dose-dependently inhibit TNF-α induced IL-1β and IL-6 secretion by fibroblast-like synoviocytes (p < 0.05, p < 0.01). To confirm the effect of Res pretreatment time on its anti-inflammatory effect, the study further compared the levels of IL-1β and IL-6 secreted by cells after Res pretreatment for 12, 24, and 48 h. As shown in Figs. 2B–D, the protective effect of Res in attenuating TNF-α induced cellular inflammatory response was enhanced with increasing pretreatment effect time, but there was almost no difference between the effect of Res pretreatment for 2 and 48 h on reducing the secretion of inflammatory factors (p > 0.05). Based on the results, 24 h was selected as the time of Res pre-intervention for subsequent experiments.

Fig. 2. Resveratrol Inhibits TNF-α Induced Inflammatory Response

A The level of IL-1β under different concentrations of Res treatment in cell supernatant. B The level of IL-1β under different times of Res treatment in cell supernatant. C The level of IL-6 under different concentrations of Res treatment in cell supernatant. D The level of IL-6 under different times of Res treatment in cell supernatant. Compared with the Control group, ^#p < 0.05, ^##p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. n = 3.

Resveratrol Upregulated Sirt1, CST mRNA and Protein Expression

As shown in Fig. 3, TNF-α injury remarkably decreased Sirt1, CST mRNA and protein expression in Model group cells (p < 0.01). Res pre-incubation for 24 h restored Sirt1, CST mRNA and protein expression in cells noticeably (p < 0.05, p < 0.01). The upregulation of Sirt1, CST mRNA and protein expression by Res was dose-dependent in the range of 100 µM, which was consistent with the tendency of Res to inhibit TNF-α induced inflammatory response (Fig. 2). It is suggested that the anti-inflammatory effect of Res in fibroblast-like synoviocytes may involve Sirt1 and CST. While 100 µM was selected as the concentration of Res pre-intervention for subsequent experiments based on the results of “Resveratrol Inhibits TNF-α Induced Inflammatory Response” and “Resveratrol Upregulated Sirt1, CST mRNA and Protein Expression.”

Fig. 3. Resveratrol Upregulates Sirt1 and CST mRNA and Protein Expression

A The expression of Sirt1 mRNA in cells. B Representative pictures of Sirt1 protein expression and protein immunoblotting in cells. C The expression of CST mRNA in cells. D Representative pictures of CST immunofluorescence in cells. Compared with the Control group, ^#p < 0.05, ^##p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. n = 3.

Sirt1 Inhibitor Attenuated the Inhibitory Effect of Resveratrol on Inflammatory Response

As shown in Fig. 4, IL-1β and IL-6 mRNA expression and secretion levels of cells were higher in the EX527 group compared with the Control group (p < 0.01). It indicates that in the absence of TNF-α injury, the Sirt1 inhibitor EX527 could directly stimulate the cells to secrete inflammatory factors, suggesting an important role of Sirt1 in the inflammatory response of fibroblast-like synoviocytes. In contrast, in the TNF-α induced inflammation model, the IL-1β and IL-6 mRNA expression and secretion levels of cells were markedly decreased in the Res group (p < 0.01), which were not obviously changed in the Res + EX527 group (p > 0.05) compared with the Model group. And the difference between the Res + EX527 group and the Res group was remarkable (p < 0.01). It indicates that Res pre-intervention can inhibit TNF-α induced inflammatory factor mRNA expression and secretion levels in fibroblast-like synoviocytes, however this effect can be abolished by Sirt1 inhibitor. The inhibition of Res on the inflammatory response in fibroblast-like synoviocytes may involve changes in Sirt1 expression or activity.

Fig. 4. EX527 Attenuate the Inhibitory Effect of Resveratrol on the Inflammatory Response

A The expression of IL-1β mRNA in cells. B The level of IL-1β in cell supernatant. C The expression of IL-6 mRNA in cells. D The level of IL-6 in cell supernatant. Compared with the Control group, ^#p < 0.05, ^##p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. Compared with the Res group, ⁺p < 0.05, ⁺⁺p < 0.01. n = 3.

Effect of Sirt1 Inhibitor on the Upregulation of Sirt1, CST Expression by Resveratrol

As shown in Figs. 5A and B, the expression of Sirt1 mRNA (p > 0.05) and protein (p < 0.05) in cells of the EX527 group was slightly decreased compared to the Control group. Combined with the effect of EX527 on cellular inflammatory factor expression (Fig. 4), it is suggested that EX527 mainly exerts its effect by inhibiting Sirt1 activity rather than its expression. In the TNF-α induced inflammation model, both the Res and the Res + EX527 groups showed upregulation of Sirt1 mRNA compared to the Model group (p < 0.01). These results indicated that Sirt1 inhibitors barely affected the upregulation of Sirt1 expression by resveratrol. In Figs. 5C and D, the expression of CST mRNA (p < 0.01) and protein in the EX527 group was incredibly decreased compared to the normal control, suggesting that EX527 can downregulate CST expression by inhibiting Sirt1 activity. The expression of CST mRNA (p < 0.01) and protein in the Res group was markedly upregulated compared to the Model group. Meanwhile, the ability of the Res + EX527 group to increase CST expression is still a large gap compared to the RES group (p < 0.01), indicating that the upregulation of CST expression by Resveratrol can be abolished by the Sirt1 inhibitor.

Fig. 5. Effect of EX527 on the Upregulation of Sirt1 and CST Expression by Resveratrol

A The expression of Sirt1 mRNA in cells. B Representative pictures of Sirt1 protein expression and protein immunoblotting in cells. C The expression of CST mRNA in cells. D Representative pictures of CST immunofluorescence in cells. Compared with the Control group, ^#p < 0.05, ^##p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. Compared with the Res group, ⁺p < 0.05, ⁺⁺p < 0.01. n = 3.

Silencing CST Attenuates the Inhibitory Effect of Resveratrol on the Inflammatory Response

In this study, the silencing efficiency of three pairs of chemically synthesized CST siRNA interfering fragments was firstly verified. CST mRNA and protein expression of cells were detected 24 h after transfection, and the fragment 1 with the highest transfection efficiency was selected according to the results of mRNA expression for subsequent experiments. The CST mRNA expression of this group was reduced by more than 70% compared with the negative siRNA control group (p < 0.01), indicating that the transfection was successful (Fig. 6).

Fig. 6. Validation of CST siRNA

A The expression of CST mRNA in cells. B Representative pictures of CST immunofluorescence in cells. Compared with Control group, ^## p < 0.01. Compared with negative siRNA group, ^△△ p < 0.01. n = 3.

As shown in Fig. 7, in the TNF-α induced inflammation model, the expression and secretion levels of IL-1β and IL-6 mRNA in the Res group were decreased (p < 0.01), consistent with the previous results. CST siRNA can greatly interfere with the regulation of cellular inflammatory factor mRNA expression and secretion levels by RES (p < 0.01). Meanwhile, neither negative siRNA interference fragment nor transfection reagent affected the anti-inflammatory effect of Res (p > 0.05), suggesting that the inhibition of TNF-α induced inflammatory factor secretion by Res involves CST, and silencing CST could abolish the protective effect of Res. Furthermore, in the absence of TNF-α stimulation, the secretion of cellular inflammatory factor was increased in the CST siRNA group. However, only the expression and secretion levels IL-6 mRNA in this group were statistically significant difference compared to normal control group (p < 0.05), which may be attributed to the lower expression levels of basal CST and inflammatory factors in normal fibroblast-like synoviocytes.

Fig. 7. CST siRNA Attenuates the Inhibitory Effect of Resveratrol on the Inflammatory Response

A The expression of IL-1β mRNA in cells. B The level of IL-1β in cell supernatant. C The expression of IL-6 mRNA in cells. D The level of IL-6 in cell supernatant. Compared with the Control group, ^# p < 0.05, ^## p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. Compared with the Res group, ⁺ p < 0.05, ⁺⁺ p < 0.01. n = 3.

Effect of Silencing CST on the Upregulation of Sirt1 Expression by Resveratrol

As shown in Fig. 8, CST silencing did not affect Sirt1 expression in normal fibroblast-like synoviocytes (p > 0.05). In the TNF-α induced inflammation model, both the Res and Res + CST siRNA groups showed a clear upregulation of Sirt1 mRNA and protein expression compared with the Model group (p < 0.01). Moreover, there was little difference in cellular Sirt1 expression between the Res and the Res + CST siRNA groups (p > 0.05), indicating that silencing the CST gene did not affect the upregulation of Sirt1 expression by resveratrol.

Fig. 8. Effect of CST siRNA on the Upregulation of Sirt1 Expression by Resveratrol

A The expression of Sirt1 mRNA expression in cells. B Representative images of Sirt1 protein expression and protein immunoblotting in cells. Compared with the Control group, ^# p < 0.05, ^## p < 0.01. Compared with the Model group, * p < 0.05, ** p < 0.01. Compared with the Res group, ⁺ p < 0.05, ⁺⁺ p < 0.01. n = 3.

DISCUSSION

Polygonum cuspidatum (Reynoutria japonica Houtt), also known as Suan-Tang-Gan, Yin-Yang-Lian, and Huo-Xue-Long, is a perennial herb in the Polygonaceae family.⁸⁾ Since ancient times, the Chinese people have used its rhizome internally or externally to relieve joint paralysis and its complications.¹⁷⁾ Now it is used as an important component of many RA clinical prescriptions such as Bi-Qing-Yin and Taibi granules and the special preparation Nanhu gelata in Chinese Traditional Medicine.^9–11) The Polygonum cuspidatum has complex chemical composition. In addition to a variety of non-specific components, the polydatin and resveratrol are the characteristic active ingredients that are abundant in Polygonum cuspidatum.¹⁸⁾ Among them, polydatin can be broken down into resveratrol in the intestinal tract through the action of glycosidases.¹⁹⁾ Therefore, we believe that resveratrol is the basis of the material action of Polygonum cuspidatum. And this may be the mystery of Polygonum cuspidatum used for prevention and treatment in RA. Several studies have confirmed the anti-inflammatory and immunosuppressive effects of polydatin and resveratrol, which can effectively improve foot swelling, reduce arthritis index and inhibit synovial inflammatory response in collagen-induced RA models.^20,21) They are considered to have greater potential in the prevention and treatment of RA.²²⁾ However, the lack of mechanistic studies has limited its further development and utilization. Therefore, this study focus on resveratrol and explored its anti-inflammatory mechanism in primary cultured rat fibroblast-like synoviocytes.

TNF-α is a predominant inflammatory mediator in RA and other immunoinflammatory diseases, and is directly involved in the synovial inflammatory response.^23,24) In this study, we isolated primary fibroblast-like synoviocytes from rat knee joint tissue and established a cellular inflammation model by stimulating with exogenous TNF-α. The results showed that resveratrol decreased TNF-α induced cellular inflammatory factor secretion in a time- and dose-dependent manner (Fig. 2), confirming its anti-inflammatory effect in fibroblast-like synoviocytes, which is consistent with the effect observed by Chen et al.¹²⁾ in an animal model study of collagen-induced arthritis. Resveratrol has been shown to exert various pharmacological activities, such as anti-inflammatory, anti-oxidant, and anti-aging, through upregulation of Sirt1 expression or activation of Sirt1.^25,26) However, whether its anti-inflammatory effects in fibroblast-like synoviocytes involve the Sirt1 pathway has not been previously reported. In our study, resveratrol dose-dependently upregulated Sirt1 mRNA and protein expression in a TNF-α induced inflammation model (Fig. 3), which was consistent with its tendency to inhibit the secretion of inflammatory factors (Fig. 2), suggesting that its anti-inflammatory effects may involve Sirt1 upregulation. EX527 is a selective inhibitor of Sirt1 that can inhibit Sirt1 activity by blocking the formation of the ribose-Sirt1 complex.²⁷⁾ In our study, EX527 only slightly altered Sirt1 mRNA and protein expression in normal fibroblast-like synoviocytes (Fig. 5), indicating that its action mainly acts through inhibition of Sirt1 activity rather than expression. EX527 barely affected the upregulation of Sirt1 expression by resveratrol (Fig. 5) but abolished its anti-inflammatory effect (Fig. 4). In the present study, the use of the inhibitor of EX527 provides antagonism of the target protein at pharmacological level, whereas knockdown or knockout produces expressed silencing of the target protein at genetic level. Functional inhibition by pharmacological inhibitor, genetic suppression or completely knockout the protein, should have similar biological effects. In different disease models, several reports have confirmed that the Sirt1 can be efficiently blocked and similar final results were observed with the use of either Sirt1 inhibitor EX527 or Sirt1 siRNA.^28,29) Thus, the changes in either Sirt1 expression or activity can mediate the inflammatory response, and that resveratrol exerts its effect mainly through upregulation of Sirt1 expression in fibroblast-like synoviocytes.

CST is a highly regarded anti-inflammatory active peptide that competitively binds to TNF-α receptors, inhibits macrophage activation and Th1 cell proliferation, reduces inflammatory factor production and promotes anti-inflammatory factor secretion.^5,30,31) Injected CST can reduce serum antibody immunoglobulin G2a (IgG2a) and pro-inflammatory factors in collagen-induced arthritis mice, improve joint swelling, inhibit inflammatory cell infiltration, and improve cartilage and bone damage.³²⁾ However, its pleiotropic nature and adverse effects, as well as the need for injectable administration, limit its further research and development as a drug. Resveratrol excitingly upregulated endogenous CST expression, inhibited TNF-α production and reduced joint swelling and cartilage destruction in an animal model of collagen-induced arthritis,¹²⁾ bringing new opportunities for CST applications. In the present study, resveratrol dose-dependently upregulated CST mRNA and protein expression in fibroblast-like synoviocytes (Fig. 2), while silencing CST expression abolished the effect of resveratrol in inhibiting TNF-α induced inflammatory factor secretion (Fig. 7), suggesting that the anti-inflammatory effect of resveratrol through upregulation of Sirt1 may involve CST. The mutual interaction between Sirt1 and CST has not been reported yet currently. In order to clarify the association and confirm the mechanism of action of resveratrol, the effect of Sirt1 inhibitor EX527 on CST expression and the effect of silencing CST on Sirt1 expression in fibroblast-like synoviocytes were investigated. The results showed that the upregulation of CST expression by resveratrol could be abolished by the Sirt1 inhibitor in the TNF-α induced inflammation model (Fig. 5), however silencing CST did not affect the upregulation of Sirt1 expression by resveratrol (Fig. 8). This suggests that Sirt1 may be an upstream gene of CST and that resveratrol achieves its anti-inflammatory effects in fibroblast-like synoviocytes by upregulating Sirt1 expression and subsequently upregulating CST.

Resveratrol is a natural active ingredient with the potential to upregulate endogenous anti-inflammatory active peptide CST expression, making it highly valuable for research and application purposes. In this study, a TNF-α induced inflammatory cell model was established using primary fibroblast-like synoviocytes from rats to investigate the mechanism of action by which resveratrol inhibits the inflammatory response. The findings suggest that resveratrol regulates endogenous CST mainly through upregulation of Sirt1 expression. Our further study is to validate the anti-inflammatory action targets and mechanism pathways of resveratrol in an animal model of rheumatoid arthritis, providing new clinical treatment strategies for RA and unique perspectives for resveratrol and the traditional Chinese medicine Polygonum cuspidatum.

Acknowledgments

This research was funded by The First-Class Discipline of Pharmaceutical Science of Hunan, and The Research Projects of Hunan Provincial Health Commission (Grant No. B2016095). The authors sincerely thank Huanghe Yu for his excellent linguistic assistance.

Conflict of Interest

The authors declare no conflict of interest.

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