南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (6): 913-921.doi: 10.12122/j.issn.1673-4254.2022.06.16

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大黄素治疗类风湿关节炎的作用机制：基于网络药理学方法

曹春浩，曾 丽，荣晓凤   

  1. 重庆医科大学附属第一医院中西医结合科，重庆 400016
  • 出版日期:2022-06-20 发布日期:2022-06-27

Therapeutic mechanism of emodin for treatment of rheumatoid arthritis: a network pharmacology-based analysis

CAO Chunhao, ZENG Li, RONG Xiaofeng   

  1. Department of Integrated Traditional Chinese and Western Medicine, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Online:2022-06-20 Published:2022-06-27

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摘要： 目的 基于网络药理学方法，运用在线数据库研究大黄素治疗类风湿关节炎的作用机制，并在成纤维样滑膜细胞细胞系中进行验证。方法 通过Targetnet、SwissTargetPrediction、Genecards、DisGeNET和OMIM数据库，分别获得大黄素作用靶点及类风湿关节炎相关基因，并构建靶点蛋白质相互作用网络（PPI），对交集基因进行Gene Ontology与KEGG通路富集分析。运用AutoDock4.2.6软件模拟分子对接。将大黄素与经TNF-α诱导的MH7A细胞共同培养，培养分组情况：空白对照组、模型组（TNF-α组，10 ng/mL）、药物干预组（TNF-α 10 ng/mL+Emodin20、40、60、80、100 μmol/L）。采用CCK-8法、细胞划痕实验及流式细胞周期实验探究大黄素对MH7A细胞增殖的抑制作用。通过Western blot观察其NF-κB通路蛋白表达情况，通过Rt-PCR实验分析 CAPS3、PTGS2（COX2）、MAPK14 基因的表达。结果 获得大黄素和类风湿关节炎交集基因32个，其中心节点为CAPS3、ESR1、MAPK14等，主要涉及NF-κB信号通路等。分子对接提示大黄素与核心靶点能较好的结合。实验结果显示，大黄素能明显抑制MH7A细胞过度增殖（P<0.05）。Western blot结果显示，TNF-α诱导的MH7A细胞NF-κB 蛋白表达水平升高（P<0.01），而予以大黄素80 μmol/L干预后IκBα、p-NF-κB表达水平均有所降低（P<0.01）。Rt-PCR结果显示，TNF-a组COX2、 P38MAPK（MAPK14）核心基因的表达量明显上调，而大黄素（80 μmol/L）处理后各组COX2、P38MAPK14的mRNA表达下降mRNA的表达下降（P<0.01），凋亡相关基因CASP3上调。结论 大黄素通过调控细胞增殖凋亡，调节NF-κB等信号通路等发挥治疗类风湿关节炎作用。

关键词: 网路药理；大黄素；类风湿关节炎；NF-κB通路

Abstract: Objective To investigate the therapeutic mechanism of emodin in the treatment of rheumatoid arthritis (RA) using a network pharmacology-based method and validate this mechanism in a fibroblast-like synovial cell line. Methods The PubChem, Targetnet, SwissTargetPrediction, Genecards, OMIM, and DisGeNET databases were searched to obtain emodin targets and RA-related genes. A protein-protein interaction (PPI) network was constructed, and GO and KEGG pathway enrichment analyses were carried out to analyze the intersection genes. AutoDock4.2.6 software was used to simulate molecular docking between emodin and its candidate targets. In a cultured fibroblast-like synovial cell line (MH7A), the effects of different concentrations of emodin on proliferation of tumor necrosis factor-α (TNF-α)-induced cells were investigated using CCK-8 assay, cell scratch experiment and flow cytometry; the changes in the expressions of nuclear factor-κB (NF-κB) pathway proteins were detected using Western blotting, and the mRNA expressions of the hub genes were examined with RT-qPCR. Results We identified 32 intersection genes of emodin and RA, and the key targets including CAPS3, ESR1, and MAPK14 involved mainly the NF-κB signaling pathway. Cell scratch experiment and flow cytometry demonstrated a strong inhibitory effect of emodin on MH7A cell proliferation. Treatment with TNF-α significantly increased the cellular expressions of the NF-κB pathway proteins, which were obviously lowered by treatment with 80 μmol/L emodin. The results of RT-qPCR showed that TNF-α treatment obviously up-regulated the expressions of the hub genes COX2 and P38MAPK, and emodin treatment significantly down-regulated the expressions of MAPK and PTGS2 and up-regulated the expression of CASP3. Conclusion The therapeutic effect of emodin on RA is mediated mainly through regulation of cell proliferation, apoptosis, and the NF-κB pathway.

Key words: network pharmacology; emodin; rheumatoid arthritis; nuclear factor-κB signaling pathway