基于网络药理及动物实验研究卢健棋教授强心汤治疗慢性心力衰竭分子机制

Molecular Mechanism of Professor Lu Jianqi's Qiangxin Decoction in the Treatment of Chronic Heart Failure Based on Network Pharmacology and Animal Experiments

目的基于网络药理学方法、分子对接技术及动物实验研究验证广西名老中医、岐黄学者卢健棋教授所创强心汤治疗CHF分子作用机制。方法首先检索TCMSP数据库与相关文献查找强心汤组成的重要化合物;通过TCMSP数据库和STITCH数据库查找强心汤组成作用靶点;运用GeneCards、DisGeNET与OMIM等数据库获取CHF的主要靶点;进一步选择Venny平台取得两者的交集靶点;使用STRING平台和Cytoscape 3.6.1构建“成分-靶点”网络与强心汤靶点-CHF靶点的PPI网络;利用DAVID 6.8数据库进行GO富集分析和KEGG通路富集分析;使用AutoDock Vina软件进行分子对接。最后构建结扎大鼠左冠状动脉前降支法造成AMI后CHF模型,采用免疫蛋白印迹法检测核心靶点蛋白表达。结果通过网络药理学研究方法得到槲皮素(Quercetin)、山柰酚(Kaempferol)、木犀草素(Luteolin)、丹参酮(Tanshinone Iia)与柚皮素(Naringenin)等185个重要活性成分,核心靶点为信号转导和转录活化因子3(STAT3)、结核分枝杆菌调节蛋白(RELA)、磷酸化蛋白激酶1(AKT1)、丝裂原活化蛋白激酶1(MAPK1)和白细胞介素6(IL-6)等100个治疗靶点,初步表明强心汤可能通过调控脂质和动脉粥样硬化、流体剪切应力和动脉粥样硬化、肿瘤坏死因子、IL-17、HIF-1、松弛素和C型凝集素受体等信号通路对细胞因子介导的信号通路、基因表达的正向调控、对缺氧的反应、对脂多糖的反应、对药物的反应等生物过程进行调节,发挥治疗CHF的作用。分子对接结果表明,强心汤组成的重要化合物与核心作用靶点均表现有强烈的结合能力;动物实验研究结果表明,强心汤成分能显著降低STAT3蛋白与MAPK1蛋白的磷酸化表达水平和IL6蛋白的表达水平(P<0.05),强心汤高剂量组略优于低剂量组。结论本研究初步阐明强心汤可以通过降低STAT3蛋白与MAPK1蛋白磷酸化和IL6蛋白表达水平起到治疗CHF的作用,也验证了强心汤有多组成、多靶点、多途径协同作用治疗CHF的特点,动物实验为临床医师治疗CHF临床用药及进一步研究提供实验理论依据。

Objective To verify the molecular mechanism of Qiangxin Decoction in treating CHF,which was created by Professor Lu Jianqi,a famous old Chinese medicine and Qihuang scholar in Guangxi,based on network pharmacological methods,molecular docking technology and animal experiments.Methods Firstly,TCMSP database and related literatures were searched to find the important compounds of Qiangxin decoction;Through TCMSP database and STITCH database,find the target of Qiangxin Tang;Get the main target points of CHF with the help of GeneCards,DisGeNET,OMIM and other databases;The Venny platform was selected to obtain the intersection target of the two;Using STRING platform and Cytoscape 3.6.1,build a"component target"network and a PPI network of Qiangxin Tang target CHF target;The DAVID 6.8 database was used for GO enrichment analysis and KEGG pathway enrichment analysis;Use AutoDock Vina software for molecular docking.Finally,the model of CHF after AMI was established by ligating the anterior descending branch of left coronary artery in rats,and the expression of core target protein was detected by Western blot.Results 185 important active components including quercetin,kaempferol,luteolin,tanshinone iia and naringenin were obtained from the analysis of network pharmacological results.The core targets were signal transduction and transcription activation factor 3(STAT3),mycobacterium tuberculosis regulatory protein(RELA),phosphorylated protein kinase 1(AKT1)100 therapeutic targets,such as mitogen activated protein kinase 1(MAPK1)and interleukin-6(IL-6),preliminarily indicate that Qiangxin decoction may regulate cytokine mediated signal pathway,positive regulation of gene expression,response to hypoxia The reaction to lipopolysaccharide,drug and other biological processes play a role in the treatment of CHF.The results of molecular docking showed that the important compounds of Qiangxin Tang had strong binding ability to the core target;The results of animal experiments showed that the components of Qiangxin decoction could significantly reduce the phosphorylation expression level of STAT3 protein and MAPK1 protein and the expression level of IL6 protein(P<0.05).The high dose group of Qiangxin Decoction was slightly better than the low dose group.Conclusion This study preliminarily clarified that Qiangxin decoction can play a role in treating CHF by reducing the phosphorylation of STAT3 protein and MAPK1 protein and the expression level of IL6 protein,and also verified that Qiangxin decoction has the characteristics of multiple components,multiple targets,and multiple ways of synergistic effect in treating CHF.Animal experiments provide experimental theoretical basis for clinical doctors to treat CHF and further research.