四逆散通过HIF-1α-VEGF信号通路抑制肝癌血管新生的作用机制研究

Mechanism of Sinisan inhibiting angiogenesis of hepatocellular carcinoma through HIF-1α-VEGF signaling pathway

基于网络药理学及动物实验,对四逆散抑制肝癌(hepatocellular carcinoma,HCC)血管新生的主要活性成分及其潜在的作用机制进行探讨。利用中药系统药理学数据库与分析平台(TCMSP)及相关文献搜集四逆散有效成分及相关蛋白质靶点,从基因数据库GeneCards中获得HCC与血管新生相关靶点,与四逆散基因靶点取交集后得到核心靶点,通过Cytoscape 3.7.2软件绘制四逆散成分-靶点图,随后利用DAVID平台进行基因本体(gene ontology,GO)富集分析、京都基因和基因组百科全书(Kyoto encyclopedia of genes and gnomes,KEGG)富集分析。最后通过动物实验对主要生物学过程进行验证。采用HE染色法观察HCC组织的病理变化;使用免疫组化染色方法明确CD31表达水平;运用Western blot检测CD31、血管内皮生长因子(vascular endothelial growth factor,VEGF)及缺氧诱导因子-1α(hypoxia-inducible factor-1alpha,HIF-1α)的表达水平;以Real-Time PCR检测VEGF mRNA表达的变化;用免疫荧光染色法检测P-VEGFR2的表达情况。网络药理学方法预测,四逆散抗HCC血管新生主要富集在HIF-1信号通路、磷脂酰肌醇-3激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路等。动物实验结果表明,四逆散可以显著降低VEGF蛋白、HIF-1α蛋白、VEGF mRNA水平及VEGFR2磷酸化水平表达(P<0.05),可能通过HIF-1α-VEGF信号通路发挥抑制HCC血管新生的作用。本研究揭示了四逆散抑制HCC血管新生多成分、多靶点、多通路协同作用的特点,为四逆散治疗HCC血管新生的进一步机制研究提供了依据。

To investigate the main active components of Sinisan in inhibiting angiogenesis of hepatocellular carcinoma(HCC)and its potential mechanism based on network pharmacology and animal experiments,we collected the active components and related protein targets of Sinisan by using TCMSP and related literatures.After intersecting HCC,angiogenesis related targets and Sinisan gene targets in GeneCards,we drew the component-target map of Sinisan by Cytoscape 3.7.2 software,and then enriched the gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)by DAVID platform.Finally,the main biology effects were verified by animal experiments.The pathological changes of HCC were observed by HE staining;the expression level of CD31 was determined by immunohistochemical staining;the expression of CD31,VEGF protein and HIF-1αprotein was detected by Western blot;the expression of VEGF mRNA was detected by Real-Time PCR;the expression of P-VEGFR2 was detected by immunofluorescence staining.Network pharmacology predicted that the anti-angiogenesis of Sinisan was mainly concentrated in HIF-1 pathway,PI3K-Akt pathway and so on.The results of animal experiments showed that Sinisan could inhibit the angiogenesis of HCC by significantly expressing VEGF mRNA and VEGFR2 phosphorylation through HIF-1α-VEGF pathway(P<0.05).This study reveals the synergistic effect of Sinisan on HCC angiogenesis with multi-components,multi-targets and multi-pathways,and further provides a basis for the study of the mechanism of Sinisan in treating HCC angiogenesis.