基于体外细胞实验和网络药理学研究乳香挥发油抗心脏肥大的作用机制

Analysis on mechanism of frankincense volatile oil in prevention and treatment of cardiac hypertrophy based on in vitro cell experiment and network pharmacology

目的:通过体外细胞实验和网络药理学方法探讨乳香挥发油抗心脏肥大的作用机制。方法:通过异丙肾上腺素(isoproterenol,ISO)诱导的H9c2心肌细胞肥大模型研究乳香挥发油抗心脏肥大的作用。通过CNKI、Pubmed、Pubchem等数据平台检索筛选并收集乳香挥发油活性成分、作用靶点及心脏肥大疾病靶点;利用String数据库和Cytoscape 3.8.0软件构建乳香挥发油抗心脏肥大蛋白互作(PPI)网络和乳香挥发油“药物-活性成分-关键靶点-疾病”网络,筛选分析乳香挥发油抗心脏肥大的关键靶点,并采用荧光定量PCR实验进行关键靶点的验证。通过DAVID数据库进行GO和KEGG富集分析。结果:体外细胞实验表明乳香挥发油可抑制ISO诱导的H9c2心肌细胞表面积增大、蛋白合成增加以及心脏肥大相关胚胎基因心房钠尿肽(ANP)、β-MHC mRNA表达上调。基于文献筛选乳香挥发油有效成分86种,抗心脏肥大靶点36个。网络分析雌激素受体1(ESR1)、内皮型一氧化氮合酶(NOS3)、前列腺素内过氧化物合酶2(PTGS2)、肿瘤坏死因子(TNF)、丝裂原活化蛋白激酶14(MAPK14)、过氧化物酶体增殖物激活受体γ(PPARG)是关键靶点,荧光定量PCR结果显示乳香挥发油抑制ISO诱导的心肌细胞ESR1、PTGS2、TNF、MAPK14 mRNA水平上调,NOS3、PPARG mRNA水平下调。GO功能富集分析主要涉及脂多糖介导的信号通路、一氧化氮生物合成过程的正调控、质膜穴样内陷、酶结合等。KEGG通路富集分析包含通路22条,主要与VEGF signaling pathway,TNF signaling pathway,Sphingolipid signaling pathway等相关。结论:乳香挥发油活性成分可能是通过作用于ESR1、NOS3、PTGS2、TNF、MAPK14、PPARG等关键靶点,调节VEGF signaling pathway,TNF signaling pathway,Sphingolipid signaling pathway等途径,影响脂多糖介导的信号通路、一氧化氮生物合成过程的正调控、质膜穴样内陷、酶结合等改善心脏肥大。

AIM:To explore the potential mechanism of frankincense volatile oil in the prevention and treatment of cardiac hypertrophy based on in vitro cell experiment and network pharmacology.METHODS:The anti-hypertrophic effect of frankincense volatile oil was investigated by isoproterenol induced H9c2 cardiomyocytes hypertrophy model.The active chemical components and targets of frankincense volatile oil and targets associated with cardiac hypertrophy were obtained by CNKI,Pubmed,Pubchem databases,etc.String database and Cytoscape 3.8.0 software were used to construct protein-protein interaction network(PPI)and a network of"drug-active component-key target-disease"of frankincense volatile oil in order to screen the key targets of frankincense volatile oil against cardiac hypertrophy.The fluorescent quantitative PCR experiments were performed to verify those key targets.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway annotation analysis of key target genes were performed using David online analysis tool.RESULTS:In vitro cell experiments showed that frankincense volatile oil significantly inhibited the isoproterenol induced increases in cardiomyocytes surface area and protein synthesis,and upregulations of ANP andβ-MHC mRNA.A total of 87 active components and 36 ingredient-disease targets of frankincense volatile oil were screened.Network analysis showed that ESR1,NOS3,PTGS2,TNF,MAPK14,and PPARG were key targets.Fluorescence quantitative PCR experiments results indicated that frankincense volatile oil inhibited isoproterenol induced upregulations of ESR1,PTGS2,TNF,and MAPK14 mRNA levels,and downregulations of NOS3,PPARG mRNA levels,respectively.In addition,the GO functional enrichment analysis showed that its biological pathways mainly included lipopolysaccharide-mediated signaling pathway,positive regulation of nitric oxide biosynthetic process,caveola,enzyme binding,etc.The KEGG pathway enrichment analysis included 22 KEGG pathways,which were closely related to VEGF signaling pathway,TNF signaling pathway,sphingolipid signaling pathway and others.CONCLUSION:The active components of frankincense volatile oil may regulate VEGF signaling pathway,TNF signaling pathway,Sphingolipid signaling pathway by acting on ESR1,NOS3,PTGS2,TNF,MAPK14 and PPARG targets,thereby affecting the regulation of lipopolysaccharide-mediated signaling pathway,positive regulation of nitric oxide biosynthetic process,caveola,and enzyme binding,and improving cardiac hypertrophy.