基于网络药理学及动物实验探讨五灵胶囊抗肝纤维化的作用机制

Mechanism of Wuling Capsules against hepatic fibrosis based on network pharmacology and animal experiments

采用网络药理学、分子对接及动物实验探究五灵胶囊抗肝纤维化的潜在作用机制。首先通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)、中医药综合数据库(Traditional Chinese Medicines Integrated Database,TCMID)和GeneCards等数据库,以及文献检索获取五灵胶囊抗肝纤维化的化学成分与作用靶点,采用STRING数据库和Cytoscape 3.9.1软件对交集靶点进行蛋白-蛋白互作(protein-protein interaction,PPI)网络分析,筛选出核心靶点,并对核心靶点进行基因本体(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,进一步构建“药物-核心成分-靶点-通路-疾病”网络,随后利用AutoDock Vina软件对核心成分与核心靶点进行分子对接,预测其抗肝纤维化的潜在作用机制。最后构建四氯化碳(CCl_(4))诱导的肝纤维化大鼠模型,观察大鼠一般体征和肝组织形态,采用苏木精-伊红(HE)及Masson染色对肝组织切片进行分析,并通过ELISA、实时荧光PCR等研究五灵胶囊对炎症因子、羟脯氨酸(HYP)水平及核心靶点的作用。筛选得到五灵胶囊化学成分445个,其对应潜在靶点3882个,与1240个肝纤维化疾病靶点相交,筛选得到TNF、IL6、INS、PIK3CA等47个核心靶点。GO功能和KEGG通路富集分析得知,核心靶点主要影响细胞刺激反应过程、代谢调节过程,涉及癌症、PI3K-Akt、MAPK等信号通路。分子对接结果显示,五灵胶囊的核心成分赤芝酸K、灵芝酸B、赤芝酸N、柴胡皂苷Q2、新隐丹参酮等与核心靶点TNF、IL6、PIK3CA均有较高的亲和力。动物实验结果显示,五灵胶囊可以减少大鼠肝脏中脂肪空泡、炎症浸润以及胶原沉积,降低炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及HYP水平,下调PI3K、Akt mRNA的表达量。结果表明,五灵胶囊可能通过调控PI3K-Akt信号通路,降低TNF-α、IL-6炎症因子水平,抑制胶原过度沉积,达到抗肝纤维化的作用。

The present study aimed to explore the underlying mechanism of Wuling Capsules in the treatment of hepatic fibrosis(HF)through network pharmacology,molecular docking,and animal experiments.Firstly,the chemical components and targets of Wuling Capsules against HF were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Traditional Chinese Medicines Integrated Database(TCMID),GeneCards,and literature retrieval.The protein-protein interaction(PPI)network analysis was carried out on the common targets by STRING database and Cytoscape 3.9.1 software,and the core targets were screened,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.Enrichment analysis was conducted on the core targets and the"drug-core component-target-pathway-disease"network was further constructed.Subsequently,molecular docking between core components and core targets was conducted using AutoDock Vina software to predict the underlying mechanism of action against HF.Finally,an HF model induced by CCl_(4)was constructed in rats,and the general signs and liver tissue morphology were observed.HE and Masson staining were used to analyze the liver tissue sections.The effects of Wuling Capsules on the levels of inflammatory factors,hydroxyproline(HYP)levels,and core targets were analyzed by ELISA,RT-PCR,etc.A total of 445 chemical components of Wuling Capsules were screened,corresponding to 3882 potential targets,intersecting with 1240 targets of HF,and 47 core targets such as TNF,IL6,INS,and PIK3CA were screened.GO and KEGG enrichment analysis showed that the core targets mainly affected the process of cell stimulation response and metabolic regulation,involving cancer,PI3K-Akt,MAPK,and other signaling pathways.Molecular docking showed that the core components of Wuling Capsules,such as lucidenic acid K,ganoderic acid B,lucidenic acid N,saikosaponin Q2,and neocryptotanshinone,had high affinities with the core targets,such as TNF,IL6 and PIK3CA.Animal experiments showed that Wuling Capsules could reduce fat vacuole,inflammatory infiltration,and collagen deposition in rat liver,decrease the levels of inflammatory cytokines TNF-α,IL-6,and HYP,and downregulated the expressions of PI3K and Akt mRNA.This study suggests that the anti-HF effect of Wuling Capsules may be achieved by regulating the PI3K-Akt signaling pathway,reducing the levels of TNF-αand IL-6 inflammatory factors,and inhibiting the excessive deposition of collagen.