网络药理学、分子对接结合实验验证探讨青白通痹胶囊治疗类风湿关节炎的作用机制

Exploring mechanism of Qingbai Tongbi capsules in treating rheumatoid arthritis based on integration of network pharmacology and molecular docking combined with experimental validation

关键词:  青白通痹胶囊  类风湿关节炎  网络药理学  MH7A细胞  炎症

Key words:  Qingbai Tongbi Capsules  Rheumatoid arthritis  Network pharmacology  MH7A cells  Inflammation

摘要:

目的 基于网络药理学结合实验验证探讨青白通痹胶囊治疗类风湿关节炎(RA)的作用机制。方法 通过TCMSP和HERB数据库筛选并确定青白通痹胶囊中的活性成分,Swiss Target Prediction数据库预测活性成分靶点。在OMIM、GeneCards、TTD、Disgenet数据库检索RA的作用靶点,将青白通痹胶囊与RA的靶点取交集。利用STRING数据库和Cytoscape 3.9.1软件构建药物-成分-靶点网络图、蛋白相互作用(PPI)网络。京都基因与基因组百科全书(KEGG)富集分析预测青白通痹胶囊治疗RA的主要信号通路,再利用分子对接软件对青白通痹胶囊主要活性成分与核心靶点进行对接。最后选取MH7A细胞进行细胞活力、划痕愈合、关键基因mRNA表达水平分析等实验,以探索青白通痹胶囊及其潜在活性成分对MH7A细胞增殖、迁移、侵袭的影响。结果 青白通痹胶囊26个有效活性成分对应靶点515个,RA靶点1890个,交集靶点199个。其中青白通痹胶囊的主要活性成分是山奈酚、甘草素、β-甘草次酸、α-甘草次酸等。青白通痹胶囊治疗RA的核心靶点包括肿瘤坏死因子(TNF)、信号转导和转录激活因子3(STAT3)、表皮生长因子受体(EGFR)、基质金属蛋白酶(MMP9)等。基因本体(GO)和KEGG富集分析确定了1055个与RA发生相关条目和166条信号通路。实验验证显示,与空白对照组相比,青白通痹胶囊、α-甘草次酸、β-甘草次酸和左旋千金藤啶碱均可抑制MH7A细胞增殖、迁移和侵袭能力,降低炎症相关基因mRNA表达水平,下调MAPK1和NF-κB基因的表达。结论 青白通痹胶囊能够通过抑制炎性信号通路MAPK-NF-κB的活化,抑制炎症反应,促进细胞凋亡,从而改善RA。

Abstract:

ABSTRACT: OBJECTIVE To explore the mechanism of Qingbai Tongbi capsules in the treatment of rheumatoid arthritis (RA) based on network pharmacology and experimental verification. METHODS The active ingredients in Qingbai Tongbi capsules were screened and determined by TCMSP and HERB databases, and the active ingredient targets were predicted by Swiss Target Prediction database. The target of RA was searched in OMIM, GeneCards, TTD and Disgenet databases, and the target of Qingbai Tongbi capsules and RA were intersected. The drug-ingredient-target network map and protein interaction (PPI) network were constructed using STRING database and Cytoscape 3.9.1 software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis predicted the main signaling pathway of Qingbai Tongbi capsules in RA treatment. Molecular docking software was used to dock the main active components of Qingbai Tongbi capsules with the core target. Finally, MH7A cells were selected for cell viability, scratch healing and mRNA expression level analysis of key genes to explore the effects of Qingbai Tongbi capsules and its potential active ingredients on the proliferation, migration and invasion of MH7A cells. RESULTS The 26 active ingredients of Qingbai Tongbi capsules correspond to 515 targets, RA targets 1890, and intersection targets 199. The main active ingredients of Qingbai Tongbi capsules are kaempferol, liquiritigenin, 18-beta-glycyrrhetinic acid, 18-alpha-glycyrrhetinic acid and so on. The core targets of Qingbai Tongbi capsules in the treatment of RA include tumor necrosis factor (TNF), signal transduction and transcriptional activator 3 (STAT3), epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP9) and so on. Gene ontology (GO) and KEGG enrichment analysis identified 1055 items related to RA occurrence and 166 signaling pathways. The experimental results showed that compared with blank control group, Qingbai Tongbi capsules, 18-alpha-glycyrrhetinic acid, 18-beta-glycyrrhetinic acid and stepholidine could inhibit the proliferation, migration and invasion of MH7A cells, reduce the mRNA expression level of inflammation-related genes, and down-regulate the expression of MAPK1 and NF-κB genes. CONCLUSION Qingbai Tongbi capsules can improve RA by inhibiting the activation of inflammatory signaling pathway MAPK - NF-κB, inhibiting inflammatory reaction and promoting apoptosis.