基于网络药理学、分子对接及动物实验验证探讨余甘子防治酒精性肝病的作用机制

Exploration on the Mechanism of Prevention and Treatment of Alcoholic Liver Disease by Phyllanthus emblica L. Based on Network Pharmacology, Molecular Docking and Animal Experiments

关键词:  余甘子  酒精性肝病  网络药理学  分子对接  PI3K/AKT信号通路

Key words:  Phyllanthus emblica L.  chronic alcoholic liver injury  network pharmacology  molecular docking  PI3K/AKT signaling pathway

摘要:

目的：基于网络药理学结合分子对接的方法探讨余甘子防治酒精性肝病(alcoholic liver disease,ALD)的作用机制,并通过动物实验进行验证。方法：通过TCMSP和Uniprot数据库获取并筛选余甘子主要有效化学成分及靶点,运用OMIM和GeneCards数据库获取ALD作用靶点,制作韦恩图(Venn)获得二者重合靶点,使用String数据库对重合靶点构建蛋白互作网络图(PPI)；以Metascape在线网站为基础对核心靶点进行GO和KEGG通路富集分析,运用Cytoscape软件构建余甘子对ALD保护作用“有效成分-靶点-通路-疾病”网络图；并通过AutoDockTools和PyMOL对关键成分和核心蛋白进行分子对接及可视化。其次,构建慢性酒精性肝损伤(chronic alcoholic liver injury,CAL)小鼠模型,通过HE染色、酶联免疫(ELISA)法、免疫组化法(IHC)、实时荧光定量(qPCR)法及蛋白质免疫印迹(WB)法来验证网络药理学和分子对接的预测结果。结果：网络药理学显示余甘子保护酒精性肝病靶点248个；GO和KEGG分析显示蛋白质结合、PI3K/AKT、TNF信号通路等过程显著富集；分子对接表明AKT1是余甘子防治ALD的关键核心靶点。在CAL小鼠模型中,余甘子冻干粉能有效降低AST、ALT、ALP、TNF-a和CYP2E1活性,下调a-SMA、PI3K、AKT的表达；升高ADH、ALDH和IL-10的表达水平；HE染色发现余甘子冻干粉可以明显减少肝脏脂质聚滴现象,肝细胞形态恢复正常；同时WB验证其可明显降低核心靶点AKT1的表达。结论：余甘子防治酒精性肝损伤的保护是通过多成分-多靶点-多通路的相互作用,其潜在的机制可能与调节PI3K/AKT信号通路的活化、抑制炎症反应的发生有关。

Abstract:

Objective: To investigate the mechanism of Phyllanthus emblica L. in the prevention and treatment of alcoholic liver disease (ALD) based on network pharmacology and molecular docking. The results were verified by animal experiments. Methods: The main effective chemical components and targets of Phyllanthus emblica L. were obtained and screened from TCMSP and Uniprot database, and ALD related targets were gained from the OMIM and GeneCards database. The Venn was used to obtain the overlapping targets of Phyllanthus emblica L. and ALD, and construct a protein interaction network (PPI) using the String database for the overlapping targets. Based on the Metascape online website, the enrichment analysis of GO and KEGG pathway was carried out on the core targets. Cytoscape was applied to construct the network map of the “active ingredients-targets-pathyway-disease” for the protective effect of Phyllanthus emblica L. on ALD. The key components and core proteins were docked and visualized by AutoDock Tools and PyMOL. Secondly, to establish the chronic alcoholic liver injury (CAL) mouse model, to verify the predictive results of network pharmacology and molecular docking through HE staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), quantitative real-time PCR (qPCR) and Western blot (WB). Results: Network pharmacology showed that there were 248 targets of Phyllanthus emblica L. protecting ALD. The enrichment of GO and KEGG analysis showed that the protein binding, PI3K/AKT, TNF signaling pathway and other processes was active. The molecular docking showed that AKT1 was the key core target of Phyllanthus emblica L. in the prevention and treatment of ALD. In the CAL mouse model, freeze-dried powder from Phyllanthus emblica L. could effectively decreased the activities of AST, ALT, ALP, TNF-a, and CYP2E1, down-regulated the expression of a-SMA, PI3K and AKT, while it improved the expression of ADH, ALDH, and IL-10. HE staining showed that freeze-dried powder from Phyllanthus emblica L. could significantly reduce the phenomenon of lipid aggregation in the liver, and the morphology of hepatocytes returned to normal. Moreover, the results of WB confirmed that it could significantly decreased the expression of the core target AKT1. Conclusion: The protection of Phyllanthus emblica L. against ALD is through the interaction of multi-component- multi-target-multi-pathway, and its underlying mechanism may be related to the activation of PI3K/AKT signaling pathway and the inhibition of inflammatory response.