正源方抗肝纤维化的网络药理学预测分析及实验验证

Mechanism of Zhengyuan Prescription in Treatment of Liver Fibrosis Based on Network Pharmacology and Experimental Verification

目的:基于网络药理学、分子对接技术预测正源方抗肝纤维化的作用机制,并进行细胞水平实验验证。方法:使用中药系统药理学数据库与分析平台(TCMSP)及文献检索获得正源方活性成分,通过PubChem、SwissTargetPrediction、Similarity Ensemble Approach数据库筛选活性成分潜在靶点,通过OMIM、GeneCards疾病数据库收集肝纤维化的相关靶点,运用Venny平台获得正源方与肝纤维化交集靶点,利用String数据库建立蛋白质-蛋白质相互作用(PPI)网络,并通过Cytoscape-CytoNCA拓扑分析筛选核心靶点,利用String数据库进行基因本体功能注释(GO)和京都基因与基因组百科全书(KEGG)通路富集分析,利用Cytoscape软件构建药物-成分-靶点-通路网络,使用AutoDock Vina软件模拟关键候选活性成分与核心基因的结合活性。建立人转化生长因子-β1(TGF-β1)诱导的人肝星状细胞LX2活化模型,噻唑蓝(MTT)比色法检测细胞活性,蛋白免疫印迹法(WB)检测磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)信号通路相关蛋白表达。结果:网络药理学筛选得到正源方活性成分145个及相关靶点1109个,肝纤维化靶点1573个,正源方抗肝纤维化潜在作用靶点415个,拓扑分析得关键靶点61个;PPI网络分析核心靶点为Akt1、血管内皮生长因子A(VEGFA)、白细胞介素-1β(IL-1β)等,GO富集分析得到1753个条目,其功能主要与细胞凋亡、细胞增殖、酶结合等有关;KEGG富集分析得到174个条目,主要涉及癌症通路、PI3K-Akt通路、细胞凋亡通路等;分子对接显示,槲皮素、木犀草素、熊果酸等与Akt1、VEGFA、Jun原癌基因(JUN)等靶点结合活性较强;细胞实验结果表明,不同剂量的正源方给药后,可以显著抑制TGF-β1诱导的LX2细胞的活化,降低磷酸化PI3K(p-PI3K)/PI3K、磷酸化Akt(p-Akt)/Akt蛋白表达,且具有质量浓度依赖性。结论:正源方可能通过多靶点、多通路发挥抗肝纤维化作用,其机制可能与抑制肝星状细胞活化、调控PI3K-Akt信号通路有关。

Objective:This study aims to investigate the mechanism of Zhengyuan prescription in the treatment of liver fibrosis based on network pharmacology,molecular docking,and in vivo cell experiments.Methods:Active ingredients of Zhengyuan prescription were obtained by TCMSP and literature search,and potential targets of the active ingredients were screened by PubChem,SwissTargetPrediction,Similarity ensemble approach,and other databases.Related targets of liver fibrosis were collected through OMIM and GeneCards disease databases,and the intersection targets of Zhengyuan prescription and liver fibrosis were obtained by the Venny 2.1.0 platform.The protein-to-protein interaction(PPI)network was established by STRING,and core targets were selected through Cytoscape-CytoNCA topological analysis.Then,the Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomics(KEGG)pathway enrichment analysis were conducted by STRING.Cytoscape 3.9.1 software was used to construct the drug,ingredient,target,and pathway network,and AutoDock Vina software was used to simulate the binding activity of key active ingredients and key genes.Finally,the TGF-β1-induced human liver astrocyte(LX2)activation model was established.Cell ability was detected by MTT,and the related protein expression of the PI3K-Akt signaling pathway was detected by Western blot(WB).Results:According to the results of network pharmacological analysis,145 active ingredients and 1109 related targets of Zhengyuan prescription,1573 targets of liver fibrosis,and 415 potential targets of Zhengyuan prescription against liver fibrosis were screened out,and 61 key targets were identified by topological analysis.The key targets of PPI network analysis were Akt1,VEGFA,and IL-1β,and 1753 items were obtained by GO enrichment analysis.The function of PPI network analysis was mainly related to cell apoptosis,cell proliferation,and enzyme binding.KEGG enrichment analysis had 174 items,mainly involving the cancer pathway,PI3K-Akt pathway,apoptosis pathway,etc.Molecular docking showed that quercetin,luteolin,and ursolic acid had strong binding activity to Akt1,VEGFA,JUN,etc.Cell experiments showed that different doses of Zhengyuan prescription can significantly inhibit TGF-β1-induced LX2 cell activation and reduce p-PI3K/PI3K and p-Akt/Akt protein expressions in a concentration-dependent manner.Conclusion:Zhengyuan prescription may play an anti-liver fibrosis role through multiple targets and pathways,and its mechanism may be related to inhibiting the activation of LX2 and regulating the PI3K-Akt signaling pathway.