来氟米特对类风湿关节炎滑膜细胞焦亡的调控作用
Regulatory effects of leflunomide on synoviocytic pyroptosis in rheumatoid arthritis
李浩 1杨豫正 2凌益 1马熙 2马武开3
作者信息
- 1. 贵州中医药大学第二附属医院风湿免疫科,贵州贵阳 550001
- 2. 贵州中医药大学第二临床医学院,贵州贵阳 550001
- 3. 贵州中医药大学第二附属医院风湿免疫科,贵州贵阳 550001;贵州中医药大学第二临床医学院,贵州贵阳 550001
- 折叠
摘要
目的:探讨来氟米特调控CASP-1/3/IL-1β信号通路抑制人类风湿关节炎滑膜成纤维细胞焦亡的作用机制.方法:组织块法培养人类风湿关节炎滑膜成纤维细胞(human rheumatoid arthritis synovial fibroblast,HFLS-RA)予来氟米特及实验相关试剂干预.HE染色和免疫组化鉴定细胞,流式细胞术检测细胞周期,ELISA、RT-qPCR和Western blot法检测信号通路中焦亡相关炎性蛋白IL-1 β、IL-18、Caspase-1、Caspase-3、NLRP3和GSDMD表达量.结果:组织块法培养的细胞鉴定为HFLS-RA.细胞周期结果,在G1期,与阳性对照组比较,来氟米特组细胞DNA生成量升高(P＜0.05);在G2、S期,与阳性对照组比较,来氟米特组细胞DNA生成量降低(P＜0.05).ELISA结果显示,与阳性对照组比较,来氟米特组IL-1β、IL-18、Caspase-1、Caspase-3的分泌量降低(P＜0.05).RT-qPCR结果显示,与阳性对照组比较,来氟米特组IL-1β、IL-18、Caspase-1、Caspase-3、NLRP3和GSDMD的mRNA表达降低(P＜0.05).Western blot结果显示,与阳性对照组比较,来氟米特组IL-1β、IL-18、Caspase-1、Caspase-3、NLRP3和GSDMD蛋白表达量降低(P＜0.05).结论:来氟米特调控CASP-1/3/IL-1β信号通路中多个焦亡相关蛋白.通过抑制中上游的NLRP3、Caspase-1合成炎小体,减少GSDMD蛋白表达而抑制通路下游IL-1β、IL-18等炎症因子生成及释放,在细胞周期G0/G1起阻滞作用,可能是来氟米特治疗RA的机制之一.
Abstract
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.
关键词
类风湿关节炎/细胞焦亡/来氟米特/滑膜成纤维细胞/CASP-1/3/IL-1β信号通路Key words
rheumatoid arthritis/pyroptosis/leflunomide/synovial fibroblast/CASP-1/3/IL-1β signaling pathway引用本文复制引用
基金项目
国家自然科学基金面上项目(82274678)
贵州中医药大学国家与省级科技创新人才团队培育项目(贵中医TD合字[2022]004号)
出版年
2024
NETL
NSTL
万方数据