桂枝甘草汤通过多靶点、多通路治疗心力衰竭:基于网络药理学方法

Therapeutic mechanism of Guizhi Gancao Decoction for heart failure:a network pharmacology-based analysis

目的基于网络药理学方法预测桂枝甘草汤治疗心力衰竭潜在作用靶点与通路。方法通过TCMSP数据库、TCMID数据库、TCM@Taiwan数据库获得桂枝甘草汤化学成分,采用SwissTargetPrediction数据库预测药物潜在靶点信息,通过用DisGeNET数据库、drugbank数据库、TTD数据库获得心力衰竭靶点,采用Venny2.1.0对药物与疾病的靶点进行交集选取,采用Uniprot数据库转换信息,采用Cytoscape构建“有效成分-靶点-疾病”网络,进行蛋白-蛋白相互作用网络(PPI),并通过网络拓扑分析得到桂枝甘草汤治疗心力衰竭的核心靶点,并将核心靶点通过Metascape数据库进行GO分析及KEGG分析。通过Western blot验证网络药理学的预测结果,根据网络药理学结果中degree值及与心力衰竭过程相关程度筛选出3个因子(PKCα、ERK1/2和BCL2),在戊巴比妥钠溶于无血清高糖培养基处理的H9C2细胞中模拟心衰缺血缺氧环境,提取心肌细胞总蛋白,测定PKCα、ERK1/2和BCL2的蛋白含量。结果通过网络药理学的方法,得到桂枝甘草汤治疗心力衰竭的潜在靶点有190个,它们主要参与循环系统过程、氮化合物的细胞反应、阳离子稳态、MAPK级联的调节等生物过程;参与调控癌症相关通路,钙信号通路、cGMP-PKG信号通路、cAMP信号通路等38条通路。体外细胞模型的Western blot验证实验结果显示,心力衰竭组PKCα和ERK1/2蛋白相对表达量均高于正常组(P<0.05),心力衰竭+桂枝甘草汤组其相对含量降低(P<0.05);心力衰竭组BCL2蛋白相对表达量均低于正常组(P<0.05),心力衰竭+桂枝甘草汤组其相对含量与心力衰竭组相比明显升高(P<0.05)。结论桂枝甘草汤可能通过作用于PRKCA、PRKCB、MAPK1、MAPK3、MAPK8等多个靶点,下调PKCα和ERK1/2因子的蛋白表达,上调BCL2因子的蛋白表达,调控癌症相关通路、钙信号通路等多个信号通路来治疗心力衰竭。

Objective To predict the targets and pathways in the therapeutic mechanism of Guizhi Gancao Decoction(GZGCD)against heart failure(HF)based on network pharmacology.Methods The chemical components of GZGCD were analyzed using the databases including TCMSP,TCMID and TCM@Taiwan,and the potential targets of GZGCD were predicted using the SwissTargetPrediction database.The targets of HF were obtained using the databases including DisGeNET,Drugbank and TTD.The intersection targets of GZGCD and HF were identified using VENNY.Uniport database was used to convert the information,and the components-targets-disease network was constructed using Cytoscape software.The Bisogene plug-in,Merge plug-in,and CytoNCA plug-in in Cytoscape software were used for protein-protein interaction(PPI)analysis to acquire the core targets.Metascape database was used for GO and KEGG analysis.The results of network pharmacology analysis were verified with Western blot analysis.Three factors(PKCα,ERK1/2 and BCL2)were screened according to the degree value of network pharmacology results and the degree of correlation with heart failure process.The pentobarbtal sodium was dissolvein H9C2 cells treated with serum-free high glucose medium to simulate the ischemic anoxic environment of heart failure.The total proteins of myocardial cells were extracted.The protein contents of PKCα,ERK1/2 and BCL2 were determined.Results We identified a total of 190 intersection targets between GZGCD and HF using Venny database,involving mainly the circulatory system process,cellular response to nitrogen compounds,cation homeostasis,and regulation of the MAPK cascade.These potential targets were also involved in 38 pathways,including the regulatory pathways in cancer,calcium signal pathway,cGMP-PKG signal pathway,and cAMP signal pathway.Western blot analysis showed that in an in vitro H9C2 cell model of HF,treatment with GZGCD downregulated PKCαand ERK1/2 expressions and upregulated BCL2 expression.Conclusion The therapeutic mechanism of GZGCD for HF involves multiple targets including PRKCA,PRKCB,MAPK1,MAPK3,and MAPK8 and multiple pathways including the regulatory pathway in cancer and the calcium signaling pathway.