Regulatory effects of leflunomide on synoviocytic pyroptosis in rheumatoid arthritis
OBJECTIVE To explore the mechanism of leflunomide regulating CASP-1/3/IL-1β signal for suppressing the pyrogenesis of synovial fibroblasts in human rheumatoid arthritis.METHODS Human rheumatoid arthritis synovial fibroblasts were cultured by tissue culture and treated with leflunomide and experimental reagents.The expressions of IL-1β,IL-18,cas-pase-1,caspase-3,NLRP3 and GSDMD in CASP-1/3/IL-1β inflammatory signaling pathway were detected by enzyme-linked immunosorbent assay(ELISA),real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot.RESULTS Cells cultured in tissue mass were verified as HFLS-RA.In phase G1,cellular DNA production rose in leflunomide group than positive group(P＜0.05).In phases G2 and S,as compared with positive control group,cellular DNA production dropped(P＜0.05).ELISA indicated that the secretions of IL-1β,IL-18,caspase-1 and caspase-3 were significantly lower in leflunomide group than those in IL-1β+caspase-3 inhibitor group.RT-qPCR showed that the expressions of NLRP3,GSDMD,caspase-1,caspase-3,IL-1β and IL-18 mRNA were significantly lower in leflunomide group than those in IL-1β+caspase-3 inhibitor group(P＜0.05).Western blot indicated that the protein expressions of IL-1β,IL-18,caspase-1,caspase-3,NLRP3 and GSDMD were significantly lower in leflunomide group than those in IL-1β+caspase-3 inhibitor group(P＜0.05).CONCLUSION In CASP-1/3/IL-1 β signaling pathway,leflunomide regulates multiple pyroptosis-related proteins through suppressing the upstream NLRP3 and caspase-1,lowering the GSDMD protein expression,blunting the generation and release of IL-1β,IL-18 and other downstream factors and arrest at phase G0/G1 cell cycle may be potential therapeutic mechanisms of leflunomide for RA.
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