基于网络药理学探讨黄芪治疗糖尿病心肌病的作用机制及初步验证研究

Study on the mechanism of Astragalus in the treatment of diabetic cardiomyopathy based on network pharmacology and its preliminary verification study

目的:通过网络药理学,探索黄芪的活性成分,挖掘黄芪治疗糖尿病心肌病的靶基因点及作用机制。基于预测结果,在糖尿病心肌病模型小鼠对关键信号通路进行验证。方法:借助中药系统药理学平台(TCMSP)检索黄芪,设定ADME参数,明确其有效活性成分及对应靶点,通过STRING数据库将蛋白名称统一转换为对应的“Gene Symbol ID”。应用DisGeNET数据库得到糖尿病心肌病发生发展的作用基因,运用STRING数据库构建药物及疾病重叠基因蛋白-蛋白相互作用网络(PPI网络),采用Cytoscape 3.6.0构建疾病-药物-靶基因网络图和通过Cytohuba插件筛选出10个最为密切的靶基因。然后,将重叠基因进行基因本体(GO)和基于京都基因与基因组百科全书(KEGG)的富集分析。动物实验采用21只昆明小鼠随机分为正常组、模型组、中药黄芪组,每组7只。对各组小鼠心肌进行HE染色对比病理形态变化,同时采用Western Blot对MAPK信号通路关键分子ERK1、p-p38进行比较。结果:黄芪包含了20个活性成分,相应的靶点188个,糖尿病心肌病的相关靶点220个,与黄芪重叠的靶点37个;将黄芪与糖尿病心肌病的共同靶点导入STRING数据库,得到重叠基因PPI网络图,共有37个节点,391条边。将PPI网络图导入到Cytoscape3.6.0软件中,采用cytoHubba插件中的MCC算法得到最为显著的前10位hub基因,分别为AKT1、TP53、CASP3、MMP9、EGF、IL-10、CXCL8、IL-1β、VEGFA、PPARG。GO功能富集分析得到生物过程(biological process,BP)条目40个,细胞组成(cellular component,CC)条目23个,分子功能(molecular function,MF)条目22个,KEGG通路富集筛选得到94条信号通路主要涉及PI3K-AKT、MAPK、HIF-1、FOXO、TNP pathway等炎症或凋亡调控途径等;动物实验显示黄芪可以改善糖尿病心肌病小鼠心肌组织的炎症状态,模型组心肌组织ERK1、p-p38蛋白表达高于正常组,采用黄芪进行干预后,ERK1、p-p38蛋白表达明显低于模型组,差异均具有统计学意义(P<0.05)。结论:黄芪治疗糖尿病心肌病具有多靶点、多成分、多通路的作用特点,可通过调控MAPK信号通路ERK1、p-p38等蛋白表达,发挥抗炎、抗氧化应激等作用。

Objective:To explore the potential active ingredients and targets of Astragalus,and also to predict the targets and mechanisms of Astragalus in the treatment of diabetic cardiomyopathy.Based on the predicted results,the key signaling path‐ways were validated in a diabetic cardiomyopathy model mouse.Methods:Compounds and targets in Astragalus were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.The protein names to corresponding"Gene Symbol ID"was convert by STRING database.We obtained targets of diabetic cardiomyopathy data from DisGeNET datas‐ets.The protein‐protein interaction network(PPI network)was established using STRING database.Cytoscape 3.6.0 was used to construct a disease‐drug‐target gene network map and to screen the 10 closest target genes by Cytohuba plug‐in.The overlapping genes were then subjected to gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)‐based enrichment analysis.Finally,the key molecules of the MAPK signaling pathway were validated by in vitro experiments.Animal experiments were performed using 21 Kunming mice randomly divided into normal group,model group,and Chinese herbal medicine Astraga‐lus group,with seven mice in each group.The myocardium of mice in each group was stained with HE to compare the pathological morphological changes,and Western Blot was also used to compare the key molecules of MAPK signaling pathway,ERK1 and p‐p38.Results:Astragalus contained 20 active ingredients with 188 corresponding targets,220 targets related to diabetic cardio‐myopathy and 37 targets acting in conjunction with Astragalus.The common targets were imported into the STRING database to obtain a PPI network graph of overlapping genes,with 37 nodes and 391 edges.The PPI network map was imported into Cyto‐scape 3.6.0 software,and the most significant top 10 hub genes were obtained using the MCC algorithm in the cytoHubba plugin,namely AKT1,TP53,CASP3,MMP9,EGF,I L⁃10,CXCL8,I L⁃1β,VEGFA,PPARG.GO functional enrichment analysis yield‐ed 40 entries for biological process(BP),23 entries for cellular component(CC),22 entries for molecular function(MF)and 94 entries for KEGG pathway enrichment screening,mainly involving PI3K‐AKT,MAPK,HIF‐1,FOXO,TNP pathway and oth‐er inflammation or apoptosis regulatory pathways.Animal experiments showed that Astragalus can improve the inflammatory state of myocardial tissue in mice with diabetic cardiomyopathy,and the expression of ERK1 and p‐p38 protein in myocardial tissue of mice in the model group was higher than that in the normal group(P<0.05,P<0.01),and after the intervention with Astraga‐lus,the expression of ERK1 and p‐p38 protein was significantly lower than that in the model group,and the difference was statisti‐cally significant(P<0.05,P<0.01).Conclusion:Astragalus has multi‐target,multi‐component and multi‐pathway action charac‐teristics in the treatment of diabetic cardiomyopathy,which can exert anti‐inflammatory and anti‐oxidative stress effects by regulat‐ing protein expression of MAPK signaling pathway ERK1,p‐p38.