南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (3): 367-374.doi: 10.12122/j.issn.1673-4254.2022.03.08

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雷公藤甲素抑制类风湿关节炎患者的成纤维样滑膜细胞的炎症和迁移： 基于circRNA 0003353/JAK2/STAT3信号通路

王 杰，刘 健，文建庭，王 馨   

  1. 安徽中医药大学第一附属医院风湿科，安徽 合肥 230031
  • 出版日期:2022-03-20 发布日期:2022-04-12

Triptolide inhibits inflammatory response and migration of fibroblast like synovial cells in rheumatoid arthritis through the circRNA 0003353/JAK2/STAT3 signaling pathway

WANG Jie, LIU Jian, WEN Jianting, WANG Xin   

  1. Department of Rheumatology and Immunology, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China
  • Online:2022-03-20 Published:2022-04-12

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摘要： 目的 探讨雷公藤甲素（TPL）通过调控环状非编码RNA（circRNA）0003353对类风湿关节炎（RA）成纤维样滑膜细胞（FLS）炎症和细胞迁移的作用机制。方法 纳入我院风湿科住院RA患者50例和正常人30例，收集外周血单个核细胞（PBMCs）及血清，检测circRNA 0003353的表达、免疫炎症指标[类风湿因子（RF）、C反应蛋白（CRP）、红细胞沉降率（ESR）、anti-CCP、IgA、IgG、IgM、C3、C4]和计算DAS28评分；采用最佳浓度10 ng/mL TPL处理RA-FLS，并转染circRNA 0003353过表达质粒。采用CCK-8法和Transwell实验检测RA-FLS细胞活力、迁移能力；ELISA法检测细胞因子IL-4、IL-6、IL-17；RT-qPCR法检测circRNA 0003353、Western blot检测p-JAK2、p-STAT3、JAK2、STAT3蛋白。结果 circRNA 0003353在RA患者PBMCs中表达升高，在协助诊断RA方面有良好效能（AUC=90.5%，P<0.001，95% CI：0.83-0.98），circRNA 0003353与ESR、RF、DAS28呈正相关（P<0.05）；TPL呈时间依赖性降低circRNA 0003353表达，抑制RA-FLS的细胞活力和迁移能力，降低促炎细胞因子IL-6、IL-17，升高抑炎细胞因子IL-4（P<0.01）；TNF-α刺激后，RA-FLS中p-JAK2/JAK2、p-STAT3/STAT3比值升高，而TPL干预后降低（P<0.01）；在TPL干预基础上，转染circRNA 0003353过表达质粒，RA-FLS中circRNA 0003353表达升高，细胞活力和迁移能力升高，IL-4降低，IL-6、IL-17升高，p-JAK2/JAK2、p-STAT3/STAT3比值升高（P<0.01）。结论 circRNA 0003353在RA-PBMCs和RA-FLS中表达均升高，TPL可通过circRNA 0003353，调控JAK2/STAT3信号通路，抑制RA-FLS炎症和细胞迁移。

关键词: 类风湿关节炎；雷公藤甲素；circRNA 0003353；JAK2/STAT3；炎症；细胞迁移

Abstract: Objective To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect. Methods We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells. Results The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5% , P<0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P<0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P<0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P<0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/JAK2 and p-STAT3/STAT3 (P<0.01). Conclusion The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.

Key words: rheumatoid arthritis; triptolide; circRNA 0003353; JAK2/STAT3; inflammatory response; cell migration