基于网络药理学及分子对接的五子衍宗丸治疗肝纤维化作用机制探讨

Exploring mechanism of Wuzi Yanzong Pills in treating hepatic fibrosis based on network pharmacology and molecular docking

目的 基于网络药理学和分子对接技术探讨补肾复方五子衍宗丸治疗肝纤维化的作用机制。方法 使用中药系统药理学数据库与分析平台(TCMSP,http://lsp.nwu.edu.cn/tcmspsearch.php)数据库预测五子衍宗丸有效成分的靶点并通过Uniprot数据库进行靶点名称标准化;通过OMIM、Gene Cards、DisGeNET数据库收集肝纤维化靶点,通过韦恩图筛选活性成分靶点和疾病靶点基因的交集,获取五子衍宗丸治疗肝纤维化的潜在靶点;运用Cytoscape 3.9.0构建有效成分-靶点-肝纤维化网络,预测核心靶点,构建基于STRING平台的蛋白质-蛋白质相互作用(PPI)网络;利用微生信平台进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析;使用AutoDockTools软件,结合PPI网络及GO功能、KEGG通路分析,将核心靶点与有效成分进行对接验证。结果 获得五子衍宗丸活性成分84个,筛选出有效成分作用靶点179个,肝纤维化靶点798个,五子衍宗丸与肝纤维化交集靶点81个,经拓扑属性分析筛选得到五子衍宗丸26个活性成分、37个治疗肝纤维化关键靶点;根据“活性成分-靶点-通路”网络图,预测槲皮素、山柰酚、苦参碱、黄豆黄素为五子衍宗丸作用于肝纤维化的重要活性成分;RAC-α丝氨酸/苏氨酸蛋白激酶(AKT1)、肿瘤坏死因子(TNF)、白细胞介素-6(IL6)、细胞肿瘤抗原p53(TP53)、血管内皮生长因子A(VEGFA)等为核心靶点。GO功能富集分析的生物学过程(BP)主要与RNA聚合酶II启动子转录的正调控、基因表达的正调控等关联;细胞组分(CC)主要与细胞外空间、细胞核等关系密切;分子功能(MF)主要与大分子复合物结合、蛋白激酶活性、RNA聚合酶II核心启动子近端序列特异性DNA结合等相关。五子衍宗丸治疗肝纤维化途径主要包括化学致癌-活性氧信号通路、JAK-STAT信号通路、PI3K-Akt信号通路、MAPK信号通路等。核心成分槲皮素、山柰酚、苦参碱、黄豆黄素与核心作用靶点AKT1、TNF、IL6、VEGFA、CASP3等对接结合稳定,分子对接初步证明关键成分自发地与多个核心蛋白结合,可对多个关键靶点进行调控。结论 通过网络药理学及分子对接初步揭示补肾复方五子衍宗丸影响凋亡过程的正负调控、氧化应激、炎症因子、血管生成等反应过程,具有多成分、多靶点、多通路等作用特点,可为临床肝纤维化治疗中重视补肾法提供参考依据。

Objective The mechanism of the kidney-tonifying formula Wuzi Yanzong Pills was explored in treatment of hepatic fibrosis by means of network pharmacology and molecular docking technology.Method The active components of Wuzi Yanzong Pills and it's corresponding targets were predicted by Using TCMSP database and the target names were standardized through the Uniprot database.The hepatic fibrosis targets were collected through OMIM,Gene Cards,and DisGeNET databases,the potential targets of Wuzi Yanzong Pills in treatment of liver fibrosis were obtained by the intersection of active component targets and disease targets selected by Venn diagram.Cytoscape 3.9.0 software was used to map the active components-intersection targets-disease network and to predict core targets.STRING platform was used to construct a PPI network.Pathway enrichment was analyzed by gene ontology(GO) function and Kyoto encyclopedia of geneses and genomes(KEGG);AutoDockTools software was used to dock the core target with active components.Result 84 active components of Wuzi Yanzong Pills and 179 potential targets of the active components were obtained,798 hepatic fibrosis targets and 81 intersection targets between the disease and Wuzi Yanzong Pills were identified.26 active components and 37 key targets for the treatment of hepatic fibrosis by Wuzi Yanzong Pills were screened by meridian topology analysis.Quercetin,kaempferol,matrine,and glycitein were important active components of Wuzi Yanzong Pills in treating hepatic fibrosis which were predicted by the "active ingredient target pathway" network.The core targets were RAC-αSerine/threonine protein kinase(AKT1),tumor necrosis factor(TNF),interleukin-6(IL6),cell tumor antigen p53(TP53),vascular endothelial growth factor A(VEGFA),etc.The biological process of GO functional enrichment analysis were mainly related to the positive regulation of RNA polymerase II promoter transcription and gene expression,the cellular components were closely related to the extracellular space,nucleus,etc.,the molecular function was mainly related to the binding of macromolecular complexes,protein kinase activity,and specific DNA binding to the proximal sequence of RNA polymerase II core promoter.The main pathways through which Wuzi Yanzong Pills treated hepatic fibrosis include the chemical carcinogenic reactive oxygen species signaling pathway,JAK-STAT signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,etc.The core components of quercetin,kaempferol,matrine,and glycitein had stable docking and binding with core targets such as AKT1,TNF,IL6,VEGFA,CASP3,etc.Molecular docking had preliminarily demonstrated that key components spontaneously bind to multiple core proteins and can regulate multiple key targets.Conclusion The mechanism of the kidney-tonifying formula Wuzi Yanzong Pills in treatment of hepatic fibrosis was preliminarily revealed that it could interfere the the process of apoptosis through positive and negative regulation,oxidative stress,inflammatory factors,angiogenesis,and other response;The kidney tonifying formula Wuzi Yanzong Pills had the characteristics of multiple components,multiple targets,and multiple pathways,which can provide a reference basis for emphasizing the kidney tonifying method in the clinical treatment of hepatic fibrosis.