应用网络药理学和分子对接技术探讨金虎通胆汤治疗胆石症的作用机制及实验验证

Network pharmacology and molecular docking to discuss the mechanism of Jinhutongdan prescription in the treatment of cholelithiasis and experimental verification

目的:基于网络药理学方法分析金虎通胆汤治疗胆石症的作用机制并进一步通过动物实验验证金虎通胆汤对活性靶点的药理作用。方法:(1)应用中药系统药理学分析平台(TCMSP)和蛋白数据库(UniPort),获得金钱草、虎杖、茵陈、香附的有效成分,并对它们的作用靶标进行研究。通过Genecards数据库和OMIM数据库挖掘胆石症相关疾病靶点,并与药物活性成分靶点进行映射,取得交集,得到金虎通胆汤治疗胆石症的共同(直接)靶点。通过Cytoscape3.9.0软件以及String数据库构建靶蛋白互作(PPI)网络及活性成分-靶点网络。利用Metascape数据库完成GO功能和KEGG通路富集分析,借助微生信数据分析绘制条形图,使用Cytoscape3.9.0软件构建共同靶点-信号通路网络。利用UCSF Chimera1.15软件和Autodock vina软件进行分子对接,选择蛋白互作网络中度值排名前6位的重要的靶点进行分子对接,验证结合活性。(2)取豚鼠48只,随机分为4组(n=12),除空白组外其余3组建立胆石症模型。其中两组分别给以阿司匹林,金虎通胆汤药液治疗后计算胆囊成石率,ELISA法检测血清TNF-α水平。结果:(1)共有23个活性成分通过23个共同靶点作用于胆囊结石,核心靶点主要包括EGFR、CCND1、TP53、EGF、IL-6等。共同靶点的KEGG通路富集分析显示肿瘤通路为主要富集靶点;人类巨细胞病毒感染信号通路、MAPK信号通路等13条信号通路均与胆石症相关。TNF与异鼠李素(isorhamnetin),EGFR与山奈酚(kaempferol),EGFR与异鼠李素(isorhamnetin),TP53与β-谷甾醇(beta-sitosterol)具有较强结合活性。(2)动物实验结果:模型组成石率高于正常对照组,具有显著性差异(P<0.05);金虎通胆汤组成石率较其他组低。与正常对照组比较,实验各组血清中TNF-α含量均增高(P<0.01);金虎通胆汤与阿司匹林组比较血清TNF-α水平较低。结论:(1)金虎通胆汤可能主要通过抑制炎症、增强免疫、抗肿瘤、细胞增殖调控、抗氧化等途径对胆囊结石起到治疗作用。(2)金虎通胆汤对模型豚鼠均可产生不同程度的治疗作用,其机制可能是通过调节TNF-α表达而抑制炎症因子、减轻炎症反应。

AIM:To analyze the mechanism of Jinhutongdan prescription in treating cholelithiasis based on network pharmacology and verify the pharmacological on the active target through animal experiments.METHODS:(1)The active ingredients of Jinhutongdan recipe were obtained by TCMSP and UniPort.The targets of cholelithiasis treated with Jinhutongdan recipe were obtained by the targets of cholelithiasis related diseases through Genecards database and OMIM database and mapping them with the targets of active ingredients of drugs.Target protein interaction(PPI)network and active ingredients-target network were constructed by Cytoscape3.9.0 software and String database.Using Metascape database to complete GO function and KEGG pathway enrichment analysis,using Cytoscape3.9.0 software to build common target-signal pathway network.We carried out molecular docking by UCSF Chimera1.15 and Autodock-vina software.Top 6 targets ranked by degree value of PPI were selected for molecular docking to verify the binding activity.(2)Forty-eight guinea pigs were randomly divided into 4 groups(n=12),and the other 3 groups were made cholelithiasis model except blank group.Aspirin group and Jinhutongdan recipe group were calculate the stone-formation rate.Serum TNF-αlevel was detected by ELISA.RESULTS:(1)23 active ingredients acted on cholelithiasis through 23 common targets,including EGFR,CCND1,TP53,EGF,IL-6,etc.The KEGG pathway enrichment analysis showed that 13 pathways were related to cholelithiasis.TNF and Isorhamnetin,EGFR and Kaempferol,EGFR and Isorhamnetin,TP53 and beta-sitosterol showed strong binding activity.(2)Animal experiment results:The lithogenesis rate of the model group significantly higher than blank group(P<0.05);The lithogenesis rate of Jinhutongdan recipe was lower than other groups.The serum TNF-αwas significantly increased in each group(P<0.01);Compared with serum TNF-αcontent in Jinhutongdan group was significantly decreased.CONCLUSION:(1)Jinhutongdan recipe may play a role in the treatment of cholelithiasis by inhibiting inflammation,enhancing immunity,anti tumor,regulating cell proliferation and antioxidation.(2)Jinhutongdan recipe has therapeutic effect on model guinea pigs,the mechanism may be in inhibits inflammatory factors and alleviates inflammatory response by regulating TNF-αexpression.