基于网络药理学预测心律康宁方抗缓慢性心律失常的作用机制及实验验证

Prediction of the mechanism and experimental verification of the anti-bradycardia effect of Xinlv Kangning prescription based on network pharmacology

目的:基于网络药理学预测心律康宁方治疗缓慢性心律失常的作用机制并初步进行细胞生物学验证。方法:通过中药分子机制的生物信息学分析工具(Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine,BATMAN-TCM)、人类基因的综合数据库(GeneCards)、在线人类孟德尔遗传数据库(Online Mendelian Inheritance in Man,OMIM)等数据库分别对心律康宁方和BA进行分析靶点预测,并将二者获得的靶点基因取交集,获取心律康宁方治疗缓慢性心律失常(Bradyarrhythmia,BA)的靶点基因,通过蛋白互作分析,获取核心靶点基因,通过基因本体论(The Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,获取关键基因和靶点通路;制备含有不同浓度的心律康宁含药血清,利用Langendorff灌流系统急性分离单个大鼠的心肌细胞,加入含药血清,初步验证预测结果。结果:药物靶点1591个,疾病靶点1886个,交集靶点467个,KEGG通路主要通过钙信号通路、心肌中环磷酸腺苷(Cyclic Adenosine Monophosphate,cAMP)信号通路、丝裂原活化的蛋白激酶(Mitogen-activated Protein Kinase,MAPK)、神经活性配体-受体相互作用传导,GO主要涉及分子功能、细胞成分、生物过程三方面。动物实验验证表明,与正常血清对照组相比,心律康宁低、中、高剂量含药血清对静息细胞胞浆Ca^(2+)平均荧光强度有显著的增强作用(P<0.05),前后差值两两比较(P<0.05),有剂量依赖性。结论:网络药理学预测结果较为可靠,心律康宁能促进心肌细胞自发的外钙内流,具有治疗BA的潜在作用。

Objective:To predict the mechanism and experimentally verify the anti-bradycardia effect of Xinlv Kangning prescription(心律康宁方)based on network pharmacology.Methods:Using Batman-TCM,GeneCards,OMIM and other databases,the analysis target prediction of Xinlv Kangning prescription and BA was carried out,and the intersection of target genes obtained from the two was obtained to obtain the target gene of Xinlv Kangning prescription for the treatment of BA.Through protein interaction analysis,the core target gene was obtained,and the GO and KEGG enrichment analysis was performed.TKy genes and target pathway were acquired;Langendorff perfusion system was used to isolate single rat cardiac myocytes,and the serum containing the drug was added to preliminarily verify the prediction results.Results:There were 1591 drug targets,1886 disease targets,and 467 intersection targets.KEGG pathway was mainly conducted through calcium signaling pathway,myocardial central adenosine phosphate signaling pathway,MAPK,and neuroactive ligand-receptor interaction.GO mainly involved molecular functions,cellular components,and biological processes.Animal experimental verification showed that compared with normal serum control group,the low,medium and high dose drug containing serum significantly enhanced the average fluorescence intensity of cytoplasmic Ca^(2+)in resting cells(P<0.05),and the difference was compared in a dose-dependent manner(P<0.05).Conclusion:The prediction results of network pharmacology are relatively reliable.Xinlv Kangning prescription can promote spontaneous exo-calcium influx of cardiomyocytes,which has a potential role in the treatment of BA.