基于网络药理学和分子对接探究连参止痢颗粒治疗猪腹泻的作用机制

Study on the Mechanism of Lianshenzhilikeli in the Treatment of Swine Diarrhea Based on Network Pharmacology and Molecular Docking

【目的】利用网络药理学和分子对接方法研究连参止痢颗粒治疗猪腹泻的作用机制。【方法】运用中药系统药理学分析平台(TCMSP)、中医药百科全书(ETCM)平台搜索连参止痢颗粒中黄连、白头翁、苦参、诃子、甘草的有效活性成分和作用靶点,利用Cytoscape 3.7.2软件绘制药物有效成分-靶点网络图;从NCBI、OMIM、GeneCards、MalaCards平台获取猪腹泻靶点,将疾病和药物交集蛋白靶点上传至STRING 11.5数据库,利用Cytoscape 3.7.2软件构建蛋白-蛋白互作(PPI)网络图,并利用微生信平台对核心靶点进行GO功能和KEGG通路富集分析。通过分子对接技术对关键有效活性成分和核心靶点进行对接验证。【结果】经筛选获得连参止痢颗粒药物的有效活性成分96个,药物作用靶点208个,疾病靶点1 785个。PPI结果显示,连参止痢颗粒作用于猪腹泻的关键靶点为Jun原癌基因(JUN)、丝裂原活化蛋白激酶14(MAPK14)、MYC原癌基因(MYC)、NFKB抑制剂α(NFKBIA)、原癌基因RELA(RELA)、胱天蛋白酶3(CASP3)、肿瘤坏死因子(TNF)、MAPK1、白细胞介素6(IL6)、雌激素受体1(ESR1)等。GO功能筛选得到生物过程6个条目,细胞组分和分子功能各2个条目。生物过程主要富集在RNA聚合酶Ⅱ启动子的转录正调控、正调控IL8的产生、正调控序列特异性DNA结合转录因子活性、细胞对脂多糖的反应、凋亡过程的负调控和RNA聚合酶Ⅱ启动子的转录调控;细胞组分主要富集在细胞核和细胞质;分子功能主要富集在RNA聚合酶Ⅱ核心启动子近端区域序列特异性DNA结合和DNA结合。KEGG通路富集分析获得53条通路,其中2条信号通路与猪腹泻密切相关。分子对接结果显示,连参止痢颗粒的关键有效活性成分与猪腹泻靶点具有良好的结合活性。【结论】连参止痢颗粒可能主要通过芒柄花黄素、芳香膜菊素、3,3′-二甲基槲皮素、松香素、硫酸奎尼丁、高丽槐素、1-甲氧基菜豆素、异甘草素等有效成分靶向JUN、RELA、MYC、NFKBIA、MAPK14、CASP3、TNF、MAPK1、IL6等靶点分子,调控沙门氏菌感染和IL17等信号通路,从而达到治疗猪腹泻的目的。

【Objective】 This study was conducted to reveal the mechanism of Lianshenzhilikeli in the treatment of swine diarrhea by network pharmacology and molecular docking.【Method】 Traditional Chinese Medicine Systems Pharmacology Database and Analysis platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM) platform were used to search for the active ingredients and targets of Coptidis Rhizoma,Pulsatilliae Radix,Sophorae flavescentis Radix,Chebulae fructus and licorice in Lianshenzhilikeli, and the network diagram of active ingredients and their targets was constructed by the Cytoscape 3.7.2 software.Swine diarrhea disease targets were obtained from NCBI,OMIM,GeneCards and MalaCards platform.The intersections of disease and ingredient targets were uploaded to the STRING 11.5 database, and the protein-protein interaction(PPI) network diagram was built by the Cytoscape 3.7.2 software.The GO function and KEGG signal pathway enrichment of the core targets in the PPI network were analyzed via microscopic letter platform.The key active ingredients and the core targets were verified by molecular docking technology.【Result】 96 active ingredients of Lianshenzhilikeli, 208 drug targets and 1 785 disease targets were obtained after screening.PPI analysis showed that the key targets of Lianshenzhilikeli directly targeting to swine diarrhea disease were Jun proto-oncogene(JUN),mitogen-activated protein kinase 14(MAPK14),MYC proto-oncogene(MYC),NFKB inhibitor alpha(NFKBIA),NF-κB p65(RELA),caspase-3(CASP3),tumor necrosis factor(TNF),MAPK1,interleukin 6(IL6),estrogen receptor 1(ESR1) and so on.Six items of biological process, two items of cellular component and two items of molecular function were obtained from GO function screening.Biological processes included positive regulation of transcription from RNA polymerase Ⅱ promoter, positive regulation of interleukin-8 production, positive regulation of sequence-specific DNA binding transcription factor activity, cellular response to lipopolysaccharide, negative regulation of apoptotic process and regulation of transcription from RNA polymerase Ⅱ promoter.Cellular components included nucleus and cytoplasm.Molecular functions included RNA polymerase Ⅱ core promoter proximal region sequence-specific DNA binding and DNA binding.A total of 53 signal pathways were obtained from KEGG pathway enrichment analysis, and two of them closely were relating to the disease.The results of molecular docking showed that the key active ingredients of Lianshenzhilikeli had excellent binding activity to the swine diarrhea disease targets.【Conclusion】 Lianshenzhilikeli might regulate Salmonella infection disease pathway and IL17 signaling pathway by targeting to JUN,RELA,MYC,NFKBIA,MAPK14,CASP3,TNF,MAPK1 and IL6 through the active ingredients, such as formononetin, 3′,7-dihydroxy-4′,6-dimethoxyisoflavone, 3,3′-dimethylquercetin, pinocembrin,(R)-(6-methoxy-4-quinolyl)-[(2 R,4 R,5 S)-5-vinylquinuclidin-2-yl]methanol, inermine, 1-methoxyphaseollidin, 2,4,4′-trihydroxychalcone, so as to achieve the purpose of treating the disease.