基于网络药理学探讨验证白藜芦醇抗NAFLD作用

Molecular Mechanism of Resveratrol Against NAFLD Based on Network Pharmacology

目的:基于网络药理学、分子对接技术预测白藜芦醇抗非酒精性脂肪性肝病(NAFLD)的分子机制,并根据结果在细颗粒物(PM2.5)染毒的肝细胞模型中进行验证。方法:首先通过中药系统药理学数据库和分析平台(TCMSP)、PubChem、DrugBank、SwissTargetPrediction数据库获取白藜芦醇相关靶点,与毒性与基因比较数据库(CTD)、疾病相关的基因与突变位点数据库(DisGeNET)、GeneCards、人类孟德尔遗传病数据库(OMIM)数据库中筛选得到的NAFLD靶点取交集,然后利用STRING 11.5平台构建交集靶点的蛋白互作网络图。根据Cytoscape 3.8.2软件制作靶点-通路图并分析筛选出核心模块及靶点,通过Metascape平台对交集靶点进行基因本体(GO)及京都基因和基因百科全书(KEGG)富集分析,并利用SYBYL-X 2.0软件对白藜芦醇与核心靶点进行分子对接分析。最后采用流式细胞术和蛋白免疫印迹法(Western blot)检测PM2.5染毒人肝细胞系(HepG2)的凋亡变化及凋亡相关蛋白的表达。结果:共获取白藜芦醇与NAFLD交集靶点115个,核心靶点主要包括肿瘤坏死因子(TNF)、B细胞淋巴瘤-2(Bcl-2)和胱天蛋白酶-3(Caspase-3)等。KEGG通路富集分析得174条通路,主要涉及细胞凋亡、TNF等通路。分子对接结果表明白藜芦醇与核心靶点Bcl-2、Caspase-3均有良好结合活性。流式细胞术以及Western blot结果显示白藜芦醇通过影响Bcl-2、Caspase-3蛋白的表达抑制PM2.5引起的肝细胞凋亡。结论:白藜芦醇抗NAFLD具有多通路、多靶点的特点,通过影响Bcl-2、Caspase-3靶点抑制肝细胞凋亡是其作用的重要途径,为后续研究白藜芦醇抗NAFLD的作用机制提供了理论依据。

Objective:To predict the molecular mechanism of resveratrol against non-alcoholic fatty liver disease(NAFLD)based on network pharmacology and molecular docking and verify the results on the liver cell model induced by PM2.5 exposure.Method:The targets of resveratrol were screened out from Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),PubChem,DrugBank,and SwissTargetPrediction,and the potential targets of NAFLD were retrieved from Comparative Toxicogenomics Database(CTD),DisGeNET,GeneCards,and Online Mendelian Inheritance in Man(OMIM).Then the common targets were obtained.STRING 11.5 was used to construct the protein-protein interaction(PPI)network of the common targets.Cytoscape 3.8.2 was used to plot the“target-pathway”network,and the core modules and key targets were selected.Metascape was adopted for Gene Ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses of common targets.SYBYL-X 2.0 was used for molecular docking of resveratrol to key targets.Finally,cell apoptosis and the expression of apoptosis-related proteins were detected by flow cytometry and Western blot in the PM2.5-exposed human liver cell line(HepG2).Result:A total of 115 common targets of resveratrol and NAFLD were obtained.The key targets included tumor necrosis factor(TNF),B-cell lymphoma-2(Bcl-2),and cysteinyl aspartate-specific protease-3(Caspase-3).As revealed by KEGG enrichment analysis,174 signaling pathways,represented by the apoptosis and TNF signaling pathways,were obtained.Molecular docking results showed that resveratrol had strong binding activities to Bcl-2 and Caspase-3.Furthermore,the results of flow cytometry and Western blot demonstrated that resveratrol inhibited cell apoptosis of PM2.5-exposed HepG2 cells by regulating the protein expression of Bcl-2 and Caspase-3.Conclusion:Resveratrol can treat NAFLD in a multi-pathway and multitarget way.It mainly inhibits liver cell apoptosis by affecting the expression of Bcl-2 and Caspase-3,which provides a theoretical basis for the follow-up research on the anti-NAFLD mechanism of resveratrol.