| Objective To observe the effect of resveratrol (Res) on in vitro TNF-a-induced proliferation and apoptosis of human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and further to investigate the effect of Res on the Bcl-2-associated death promoter (BAD) for the mechanism.Methods Synovial tisssues were obtained from patients with active RA according to the revised criteria of the American College of Rheumatology,who were undergoing knee joint replacement. RA FLS were stimulated with TNF-α(10ng.ml-1) for 24h in the presence or absence of resveratrol.The inhibition rate of RA FLS was examined by MTT assay. Cell cycle and the amount of apoptotic cells was measured by flow cytometry. Phosphorylated BAD protein expression was measured by western blot.Results In the lower concentration 12.5μM,25μM) of Res group, TNF-α-induced proliferation,cell phase and the apoptosis ratio of RA FLS have no obvious effections compared to TNF-a group (p>0.05), However in higher concentration(50μM,100μM,200μM), the living cells measured by MTT dose and time-dependently reduced, and the fraction of living cells in the S-phase and G2/M-phase decreased respectively, while that in G1-phase increased, the difference was statistically significant compared with the TNF-a group(P< 0.05).Flow cytometry demonstrated that the apoptosis rate increased in a dose-dependent manner among the higher concentration of Res groups. Higher concentration Res inhibited dose-dependently TNF-α-induced phosphorylation of BAD in RA FLS.Conclusions Higher concentration (50-200MM) Res can inhibit TNF-a-induced proliferation and induce apoptosis of RA FLS through blocking cell cycle and inhibition of BAD phosphorylation. Res may provide a new therapeutic approach in the treatment of RA.Objective To Observe the inhibition of Res on TNF-a-induced production of mainly inflammatory cytokines such as IL-1β,MMP3 in RA FLS, and further to explore the anti-inflammatory effects of Res from the perspective of inflammatory cytokines.Methods The levels of IL-1β,MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay (ELISA). Messenger RNA expression of IL-1βand MMP-3 in RA FLS induced by TNF-a were analyzed using a reverse transcription-polymerase chain reaction (RT-PCR). Western-blot analysis was used to detect the expression of IL-1βand MMP-3 in RA FLS intervened by Res.Results After stimulating with TNF-a for 24h, the levels of IL-1βMMP-3 sharply increased in cultural supernatants of RA FLS, respectively(436.235±79.721)pg.ml-1, (89.950±14.458)ng.ml-1, contrast to blank group(P<0.05). Res inhibited TNF-a-induced production of IL-1βand MMP-3 on RA FLS in a dose-dependent manner. Res at concentration of 12.5μM can immediately inhibited the mRNAs expression of IL-1βand MMP-3, while going to the concentration of 100,200μM, inhibitions of Res reached a peak. The Western-blot analysis showed that Res inhibited TNF-α-induced proteins expression of IL-1βand MMP-3 on RA FLS in a dose-dependent manner.Conclusions Res inhibited TNF-α-induced secretion of IL-1βand MMP-3 in RA FLS on both the mRNA and the protein level. Res plays an anti-inflammatory role via preventing cytokines production.Objective To evaluate the role of PI3K/Akt signaling pathway in TNF-a-induced proliferation and production of proinflammatory cytokines on RA FLS, and the modulation of Res on PI3K/Akt signaling pathway, further to exploring the anti-inflammatory and anti-proliferative effects of Res for its molecular mechanism.Methods Indirect immunofluorescence staining was used to detect the expression of phosphorylated Akt among RA FLS groups. The proteins expression of phosphorylated BAD,IL-1β,MMP-3,phosphorylated Akt and Akt were examined by Western-blot method.Results Immunofluorescence staining showed that the expression of phosphorylated Akt increased significantly in TNF-αgroup, while there were no obvious expression of phosphorylated Akt both in blank group and PI3K inhibitor LY294002 group. However lower expression of phosphorylated Akt was seen in 50μMRes group compared with TNF-αgroup. The Western-blot method showed the PI3K inhibitor LY294002 decreased the proteins expression of TNF-α-induced phosphorylated BAD and IL-1βMMP-3. Higher concentration Res (50-200μM) also decreased significantly the expression of phosphorylated Akt dose-dependently.Conclusions Activation of PI3K/Akt signaling pathway exists in TNF-α-induced proliferation and production of IL-1β,MMP3 on RA FLS, which is hampered by LY294002.Res can reduce the expression of phosphorylated BAD and IL-1β,MMP-3 through inhibition of Akt phosphorylation. Res exerts an anti-proliferative and anti-inflammatory effects in TNF-α-induced RA FLS, indicating Res may have good clinical application prospect in the treatment of rheumatoid arthritis.
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