基于网络药理学和动物实验探讨青蒿鳖甲汤抗血管新生的作用机制

目的:基于网络药理学预测青蒿鳖甲汤抗血管新生的潜在分子机制，并用Lewis肺癌小鼠移植瘤模型对STAT3/VEGF信号通路进行实验验证。方法:使用TCMSP、BATMAN-TCM数据库及文献筛选青蒿鳖甲汤的有效成分，利用Swiss Target Prediction平台预测成分靶点基因。GeneCards、OMIM、TTD等3个数据库检索血管新生靶点基因，与中药成分靶点基因匹配后，使用STRING数据库和Cytoscape 3.7.1软件进行蛋白质间相互作用（protein-protein interaction,PPI）分析，筛选关键靶点基因；Metascape平台对关键靶点基因进行基因本体（gene ontology,GO）功能富集分析及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析。在动物实验中，C57BL/6J雄性小鼠接种Lewis肺癌细胞，建立皮下移植瘤模型，分为模型组，青蒿鳖甲汤高（16.0 g/kg）、中（8.0 g/kg）、低（4.0 g/kg）剂量组，贝伐株单抗组（15 mg/kg），分别给予相应剂量青蒿鳖甲汤灌胃或贝伐株单抗腹腔注射。14天后处死，取皮下瘤组织，称量瘤重，计算瘤体积和抑瘤率。免疫组化法检测血管标志物CD31表达情况，计算微血管密度（microvascular density,MVD）；HE染色法观察瘤组织病理形态；Western blot法检测pSTAT3、STAT3及VEGF蛋白表达水平。结果:获取青蒿鳖甲汤有效成分61个及靶点基因523个，血管新生相关靶点基因1 229个，交集靶点基因176个，关键靶点基因22个，其中VEGF是青蒿鳖甲汤发挥抗血管新生作用的首位关键靶点基因，信号通路以癌症相关信号通路为主，如HIF-1、VEGF、JAK-STAT等。动物实验结果表明，与模型组相比，青蒿鳖甲汤中、高剂量组瘤重、瘤体积均显著降低（P＜0.05或P＜0.01），高剂量组MVD降低（P＜0.01）；各给药组肿瘤细胞体积缩小，核固缩、碎裂现象多见，可见不同程度的坏死区及出血；高剂量组VEGF、pSTAT3蛋白表达水平及pSTAT3/STAT3水平均显著降低（P＜0.05），中、低剂量组VEGF、pSTAT3蛋白水平虽有降低趋势，但差异无统计学意义。结论:青蒿鳖甲汤可以通过多成分-靶点-通路抑制血管新生，其作用机制可能与抑制STAT3/VEGF信号通路有关。

Mechanism of Qinghao Biejia Decoction in treatment of angiogenesis based on network pharmacology and animal experimental verification

Objective:To explore the underlying molecular mechanism of Qinghao Biejia Decoction on angiogenesis based on network pharmacology,and to validate the STAT3/VEGF pathway in Lewis lung carcinoma mouse graft tumor model.Methods:The main active ingredients of Qinghao Biejia Decoction were selected from TCMSP,BATMAN-TCM and literatures,and the target genes of ingredients were predicted by Swiss Target Prediction platform.The target genes related to angiogenesis were collected by three disease databases such as GeneCards,OMIM and TTD.After taking the intersection,STRING database and Cytoscape 3.7.1 software were used to construct protein-protein interaction (PPI) network and select key target genes.The Metascape platform was used to perform gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis for hub genes.In animal experiment,C57BL/6J male mice were seeded with Lewis lung carcinoma cells to establish a subcutaneous tumor graft model.Tumor-bearing mice were randomly divided into Control group,Qinghao Biejia Decoction high-dose (16.0 g/kg),medium-dose (8.0 g/kg),low-dose (4.0 g/kg) group and bevacizumab (15 mg/kg) group.The administration and model groups were infused with Qinghao Biejia Decoction or intraperitoneal injection with bevacizumab.After the end of the drug administration,the subcutaneous tumor tissues were collected.The tumor weight was assessed,and the tumor volume and tumor-inhibition rate were calculated.The expression of endothelial cell marker CD31 was measured by immunohistochemistry,and the microvascular density (MVD) was calculated.Tumor histopathological change was observed by HE staining,and protein expression levels of pSTAT3,STAT3 and VEGF were detected by Western blot.Results:A total of 61 active ingredients and 523 drug-related target genes,1 229 angiogenesis-related target genes,176 intersection target genes and 22 key target genes were obtained.VEGF was the first key target gene of the antiangiogenic effect exerting by Qinghao Biejia Decoction,and the signaling pathways were mainly cancer-related signaling pathways,such as HIF-1,VEGF and JAK-STAT.Animal experiments showed that compared with the Control group,the tumor weight and tumor volume in Qinghao Biejia Decoction high-dose and medium-dose group were decreased (P＜0.05，P＜0.01),and MVD in high-dose group was decreased (P＜0.01).The tumor cells shrinkage,pyknosis,karyorrhexis,hemorrhage and necrosis were more seen in administration group.The expression levels of VEGF,pSTAT3 protein and the proportion of pSTAT3 to total STAT3 were significantly reduced in high-dose group (P＜0.05).Although the expression levels of VEGF and pSTAT3 proteins in the medium-dose and low-dose group showed a downward trend,the difference was not statistically significant.Conclusion:Qinghao Biejia Decoction can inhibit angiogenesis through multi-component-target-pathway,and the underlying mechanism may be related to the inhibition of STAT3/VEGF signaling.