网络药理学及分子对接初步探讨毛蕊花糖苷抗急性肺损伤的作用及其机制

Preliminary exploration of the effect and mechanism of verbascoside against acute lung injury by network pharmacology and molecular docking

目的 利用网络药理学及分子对接方法初步探讨毛蕊花糖苷抗急性肺损伤(ALI)的相关分子机制。方法 利用Pubchem数据库获取毛蕊花糖苷2D结构,通过Pharmmapper数据库与SwissTargetPrediction数据库获取毛蕊花糖苷的作用靶点,通过GeneCards、OMIM、DisGeNET等数据库获取ALI的作用靶点,将毛蕊花糖苷与ALI的作用靶点交叉以获得共同作用靶点。采用String数据库及Cytoscape软件构建潜在靶点蛋白质相互作用(PPI)网络,并将交集靶点导入DAVID数据库,对基因本体(GO)功能与KEGG通路及通路靶点基因进行富集分析,利用Autodock vina软件对毛蕊花糖苷与核心靶点进行分子对接,采用实时荧光定量PCR(RT-qPCR)验证肺上皮细胞(BEA52B细胞)中核心基因mRNA表达水平,Western blotting检测相关蛋白表达情况。结果 筛选得到毛蕊花糖苷抗ALI的靶基因150个,毛蕊花糖苷抗ALI的关键靶点主要涉及PI3K-Akt信号通路、MAPK信号通路、Rap1信号通路等。毛蕊花糖苷与核心靶点分子对接良好。RT-qPCR检测结果显示,与模型组比较,毛蕊花糖苷作用后BEAS-2B细胞中HSP90AA1、ALB、TP53、TNF、HRAS、INS mRNA表达水平下降(P<0.05);Western blotting检测结果显示,与模型组比较,毛蕊花糖苷干预可明显降低p-Akt、p-p38、p-ERK蛋白的表达(P<0.05)。结论 毛蕊花糖苷可能通过抑制MAPK、Rap1和PI3K/Akt信号通路而发挥抗ALI的作用。

Objective To investigate the molecular mechanism of verbascoside against acute lung injury(ALI)by network pharmacology and molecular docking methods,and to validate the findings experimentally.Methods The 2D structure of verbascoside was obtained from the Pubchem database.Active ingredient targets of verbascoside were acquired from Pharmmapper database and Swiss Target Prediction database.Active component targets of ALI were acquired from datebase such as Gene Cards,OMIM,and DisGeNET.Common targets between verbascoside and ALI were determined by overlapping these sets.PPI network for potential targets was constructed using String database and Cytoscape software.The intersection targets were imported into the DAVID database for enrichment analysis of GO biological processes,KEGG signaling pathway and the pathway target genes.Molecular docking between verbascoside and core targets was performed using Autodock vina software.The mRNA expression level of core genes was validated using real-time quantitative PCR(RT-qPCR),and the expression of related proteins was detected using Western blotting.Results A total of 150 target genes of verbascoside against ALI were screened,and the key targets of verbascoside against ALI mainly involve pathways such as Rap1 signaling pathway,PI3K-Akt signaling pathway and MAPK signaling pathway.Verbascoside docked well with the core target molecules.RT-qPCR results showed that,compared with the control group,the mRNA expression levels of HSP90AA1,ALB,TP53,TNF,INS,and HRAS were significantly decreased in cells after the effect of verbascoside(P<0.05);Western blotting indicated that,compared with the model group,verbascoside treatment significantly reduced the expression of p-Akt,p-p38,and p-ERK proteins(P<0.05).Conclusion Verbascoside could inhibit MAPK,Rap1 and PI3K/Akt signaling pathways to exert its anti-ALI effects.