南蛇藤素干预NF-κB/MMP-9通路抑制IL-17A诱导类风湿关节炎滑膜细胞侵袭的研究

【作者】 李国青； 张育； 刘丹； 钱亚云； 张华； 郭试瑜； 砂川正隆； 久光正； 刘延庆；

【机构】 扬州大学临床医学院风湿免疫科； 扬州大学医学院中西医结合系； 日本昭和大学医学部第一生理学教室；

【摘要】 目的:为了探讨中药南蛇藤中活性成分南蛇藤素(celastrol)抗类风湿关节炎(rheumatoid arthritis,RA)滑膜细胞侵袭的机制。方法:体外原代培养人RA滑膜成纤维细胞(fibroblast-like synoviocytes,FLSs),使用MTT和流式细胞仪分析细胞周期方法确定celastrol对RA-FLS的非细胞毒浓度为实验浓度,观察其对(interleukin-17A,IL-17A)刺激的RA-FLS迁徙及侵袭能力及基质金属蛋白酶(matrix metalloproteinase,MMP)2、9表达的影响,利用RNA干扰、荧光报告酶、染色质共沉淀等方法观察不同浓度的celastrol对NF-κB信号通路相关分子、NF-κB-DNA结合能力及NF-κB调控MMPs的影响及机制。结果:非细胞毒浓度的celastrol(0.05μM,0.1μM,0.2μM)可呈浓度依赖性的抑制RA-FLS的迁徙和侵袭能力;其抗FLS侵袭与其抑制MMP-9的表达和活性相关,进一步研究发现celastrol对MMP-9转录活性的抑制与NF-κB通路活化相关,celastrol可抑制IκBα的磷酸化和降解,下调NF-κB p-65的核转位,抑制NF-κB-DNA与MMP-9启动子的结合。结论:非细胞毒浓度的celastrol可通过抑制NF-κB信号通路活化及转导,下调其下游MMP-9的转录活性,抑制IL-17A诱导的RA-FLS侵袭。更多还原

【Abstract】 Objective:Interleukin-17A(IL-17A)-induced migration and invasion of fibroblast-like synoviocytes(FLSs) is critical for the pathogenesis of rheumatoid arthritis(RA).To examine the effect and molecular mechanisms of celastrol on the migration and invasion of RA-FLSs.Methods: In this study,we investigated the effect of celastrol on the migration and invasion of human RA-FLS.Results:Results showed that treatment of RA-FLSs with celastrol suppressed the IL-17A-induced migration and invasion abilities of the cells.In addition,celastrol inhibited IL-17A-induced matrix metalloproteinase(MMP)-9 mRNA and protein expression,and the proteolytic activity of MMP-9 in RA-FLSs.Furthermore,our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of nuclear factor-κR (NF-κB) in the MMP-9 promoter,and inhibited IκBαphosphorylation and nuclear translocation of NF-κB.Conclusion:These results indicate that celastrol inhibits NF-κB -mediated MMP-9 expression,resulting in suppression of RA-FLS invasion and migration that is induced by IL-17A. Celastrol is a potential agent for clinical use as an adjunct with conventional drugs for the treatment of RA in humans.更多还原