南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1473-1483.doi: 10.12122/j.issn.1673-4254.2021.10.05

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苗药验方四大血对人类风湿性关节炎成纤维样滑膜细胞凋亡和焦亡的影响

吴 宁，袁桃花，姬进忠，程 瑶，李明义，梁 玺，孙见飞，刘 华，吴昌学   

  1. 贵州医科大学基础医学院，临床医学院，贵州 贵阳 550025；贵州医科大学医学分子生物学重点实验室，贵 州 贵阳 550004
  • 出版日期:2021-10-20 发布日期:2021-11-11

Effects of Sidaxue, a Miao ethnomedicine recipe, on apoptosis and pyrolysis of human fibroblast-like synovial cells in rheumatoid arthritis

WU Ning, YUAN Taohua, JI Jinzhong, CHENG Yao, LI Mingyi, LIANG Xi, SUN Jianfei, LIU Hua, WU Changxue   

  1. College of Basic Medical Sciences, College of Clinical Medicine, Guizhou Medical University, Guiyang 550025, China; Key Laboratory of Molecular Biology, Guizhou Medical University, Guiyang 550004, China
  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨苗药验方四大血（SX）对人类风湿性关节炎（RA）成纤维样滑膜细胞（FLSs）凋亡和焦亡的影响。方法 数据库检索RA及凋亡、焦亡相关靶蛋白；韦恩软件分析获得与RA相关凋亡及焦亡蛋白；RA-凋亡-焦亡靶蛋白互作（PPI）网络构建以获取RA中凋亡和焦亡重要靶蛋白；Autodock vina软件进行分子对接验证SX主要活性成分与凋亡和焦亡相关蛋白的结合能力，并通过PyMoL软件进行可视化。人源第2代RA-FLSs MH7A细胞检测SX对MH7A细胞抑制率后分为空白对照组、雷公藤多甘片阳性对照组（TGT组，5 mg/L）及 5、10、20、40 mg/L SX 组，采用 Wound- healing 实验、Transwell 法、ELISA 法及Western blot法分别检测MH7A细胞的迁移和侵袭能力、凋亡和焦亡相关蛋白。结果 获得RA相关凋亡靶蛋白9个、焦亡靶蛋白15个、RA-凋亡-焦亡重叠靶蛋白4个，SX主要活性成分与TNF-α、Fas、Bax等靶蛋白均具有较高亲和力；与空白组相比，给药处理24、48、72 h，随着SX浓度增加，对MH7A细胞抑制率增加（P<0.05），药物作用6、12、24 h，同一时间点内MH7A细胞划痕修复率随SX浓度增加而减少（P<0.05）；药物作用24 h，20、40 mg/L SX组MH7A细胞侵袭个数减少（P<0.05）；20、40 mg/L SX组和TGT组TNF-α、IL-1β、IL-18的表达水平均下降（P<0.05）；20、40 mg/L SX组Bax/Bcl-2比值均增加（P<0.05）；5、40 mg/L SX组Caspase-1表达量均减少（P<0.05）；20、40 mg/L SX组Fas和FasL表达量均增加（P<0.05）。结论 SX可诱导MH7A细胞凋亡、抑制其焦亡，减缓炎症反应，其作用机制可能与下调TNF-α、IL-1β、IL-18细胞因子水平，上调Bax、Fas、FasL，抑制Bcl-2和Caspase-1蛋白表达有关。

关键词: 苗药方四大血；类风湿性关节炎；细胞凋亡；细胞焦亡；数据挖掘；分子对接

Abstract: Objective To investigate the effects of Sidaxue (SX), a recipe in Miao ethnomedicine, on apoptosis and pyrolysis of human fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS). Methods The target proteins related with RA and those involved in cell apoptosis and pyroptosis were searched in different online databases, and Venny software was used to obtain apoptosis- and pyroptosis-related proteins in RA. RA-apoptosis-pyroptosis protein interaction (PPI) network was constructed to identity the key target proteins related with apoptosis and pyroptosis in RA. Autodock vina software was used to perform molecular docking to verify the binding ability of the main active ingredients in SX with the apoptosis- and pyroptosis-related proteins. In the cell experiment, MH7A cells were treated with 5 mg/L TGT (positive control) or 5, 10, 20, and 40 mg/L SX, and the changes in cell migration and invasion abilities and expressions of apoptosis- and pyroptosis-related proteins were examined using wound healing assay, Transwell assay, ELISA and Western blotting. Results We identified 9 RA-related apoptotic target proteins, 15 RA-related pyroptosis target proteins, and 4 overlapping target proteins related with RA, apoptosis and pyroptosis. The main active ingredients in SX had a high affinity with the target proteins including TNF-α, Fas, and Bax. In MH7A cells, SX treatment concentration-dependently increased the cell inhibition rate at 24, 48 and 72 h (P<0.05), and significantly lowered the cell migration ability at 6, 12 and 24 h (P<0.05); treatment with 20 and 40 mg/L SX for 24 h obviously suppressed MH7A cell invasion (P<0.05). SX treatment (20 and 40 mg/L) and TGT treatment both significantly lowered the expression levels of TNF-α, IL-1β, and IL-18 in the cells (P<0.05). The Bax/Bcl-2 ratio and Fas and FasL expressions were increased significantly in cells treated with 20 and 40 mg/L SX (P<0.05), and caspase-1 expression was decreased significantly in cells treated with 5 and 40 mg/L SX (P<0.05). Conclusion SX can induce apoptosis and pyroptosison in RA-FLSs possibly by down-regulating the expressions of TNF-α, IL-1β and IL-18, up-regulating the expressions of Bax, Fas, and FasL, and inhibiting Bcl-2 and caspase-1 protein expressions.

Key words: Miao ehtnomedicine recipe Sidaxue; rheumatoid arthritis; apoptosis; pyroptosis; data mining; molecular docking