西洋参抗心脏肥大的网络药理学分析和实验验证研究

Network Pharmacological Analysis and Experimental Verification of Panax Quinquefolius for Cardiac Hypertrophy

目的:通过网络药理学探讨西洋参多成分、多靶点、多通路的抗心脏肥大作用机制,并选取关键靶点进行细胞实验验证。方法:通过中药系统药理学数据库与分析平台(TCMSP)、中医药百科全书(ETCM)、有机小分子生物活性数据库(PubChem)、多靶点定量构效关系的靶点预测平台数据库(Targetnet)、小分子药物靶点预测在线平台(Swiss Target Prediction)等数据库及参考文献筛选西洋参活性成分及作用靶点。通过人类孟德尔遗传数据库(OMIM)、药物靶标数据库(TTD)及基因名片数据库(GeneCards)筛选心脏肥大相关靶点。将药物的作用靶点与疾病靶点进行交集,获得药物-疾病共同靶点,利用STRING数据库构建PPI网络图,并借助Cytoscape 3.8.0软件进行可视化分析,采用注释、可视化和综合发现(DAVID)数据库对重要靶点进行基因本体(GO)功能与京都基因与基因组百科全书(KEGG)通路富集分析。进一步采用异丙肾上腺素诱导的H9c2心肌细胞肥大模型,观察西洋参抗心肌细胞肥大作用,逆转录聚合酶链式反应(qRT-PCR)法检测关键靶点的基因表达。结果:从西洋参中筛选出42个活性成分,能够作用于41个心脏肥大相关靶点,其中血管内皮生长因子A(VEGFA)、丝氨酸/苏氨酸蛋白激酶1(AKT1)、表皮生长因子受体(EGFR)、肿瘤坏死因子(TNF)、丝裂原活化蛋白激酶1(MAPK1)等21个靶点是关键靶点,主要涉及一氧化氮合成过程的正调控、脂多糖介导的信号通路、肽基-丝氨酸磷酸化的正调控,参与调节癌症蛋白多糖、血管内皮生长因子(VEGF)信号通路、缺氧诱导因子-1(HIF-1)信号通路等。进一步细胞实验结果表明,西洋参100.0μg/mL可抑制异丙肾上腺素10μmol/L诱导的心肌细胞肥大(P<0.05)。在异丙肾上腺素诱导的心肌细胞肥大模型中,VEGFA、AKT1、EGFR、TNF、MAPK1 mRNA表达上调(P<0.05),西洋参可明显抑制上述关键靶点基因表达上调(P<0.05)。结论:西洋参抗心脏肥大的作用机制可能与下调VEGFA、AKT1、EGFR、TNF、MAPK1等多个靶点的基因表达有关。

Objective:To explore the mechanism of Panax quinquefolius in cardiac hypertrophy by network pharmacology,and to verify the key targets in cardiomyocytes hypertrophy model in vitro.Methods:The traditional Chinese medicine system pharmacology database(TCMSP),the Encyclopedia of Traditional Chinese Medicine(ETCM),PubChem,Targetnet,Swiss Target Prediction databases,and references were used to obtain the active components and targets of Panax quinquefolius.The targets related to cardiac hypertrophy were searched through Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),and the human gene database(GeneCards).The common targets were obtained by intersecting drug targets with disease targets.In order to screen the key common targets,STRING database and Cytoscape software were used to analyze the protein-protein interaction of common targets.DAVID database was used to conduct gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis on the common targets to obtain the relevant biological processes and signal pathways.The H9c2 cardiomyocytes hypertrophy model induced by isoproterenol was further used to observe the anti-cardiac hypertrophy effect of Panax quinquefolius,and real-time quantitative polymerase chain reaction(qRT-PCR)was performed to detect the gene expression of key targets.Results:Forty-two active components were screened from Panax quinquefolius,which could act on 41 targets related to cardiac hypertrophy.Among them,21 targets,including vascular endothelial growth factor A(VEGFA),serine/threonine protein kinase1(AKT1),epidermal growth factor receptor(EGFR),tumor necrosis factor(TNF),and mitogen activated protein kinase 1(MAPK1)were the key targets.They were mainly involved in nitric oxide synthesis,lipopolysaccharide-mediated signaling pathway,myocardial contraction,cell proliferation and migration,and other biological processes,as well as the regulation of cancer proteoglycan,vascular endothelial growth factor(VEGF)signaling pathway and hypoxia-induced-factor-1 signaling pathway(HIF-1),etc.Cell experimental results showed that Panax quinquefolius 100.0μg/mL could inhibite 10μmol/L of isoproterenol induced cardiomyocytes hypertrophy(P<0.05).Panax quinquefolius inhibited isoproterenol induced significant upregulations of genes including VEGFA,AKT1,EGFR,TNF,and MAPK1(P<0.05).Conclusion:The anti-cardiac hypertrophy mechanism of Panax quinquefolius may be related to the gene expression of multiple targets including VEGFA,AKT1,EGFR,TNF,and MAPK1.