基于网络药理学研究板蓝根抑菌活性成分及其作用机制

Network Pharmacology Study on the Antibacterial Activity Components and Mechanism of Isatisindigotica

旨在基于网络药理学研究板蓝根的主要抑菌药效成分及其作用机理。利用中药系统药理学在线分析数据库(TCMSP)筛选出板蓝根药效成分及其潜在作用靶点,通过OMIM和GeneCards数据库以“抑菌”为关键词搜索相关靶点。通过Venny平台获取板蓝根和抑菌的交集靶点。通过STRING数据库结合Cytoscape软件构建PPI网络图并筛选关键靶点。通过DAVID数据库进行GO功能富集和KEGG通路富集分析。通过Autodock Tools 1.5.7软件将关键药效成分和抑菌靶点进行分子对接。通过菌落计数法和SDS-PAGE法验证关键药效成分抑菌作用和作用靶点。结果显示:共筛选到药效成分33个,药物靶点61个,抑菌靶点2192个,药物与抑菌作用交集靶点32个。蛋白互作网络分析发现,TNF、TP53、CASP3、JUN、ESR1、PTGS2等30个蛋白可能是板蓝根抑菌作用的关键靶点。GO功能富集分析得到126个条目,包括生物过程条目86个(如对雌二醇的反应、凋亡信号通路、神经元凋亡过程的正调控),细胞组分条目11个(如细胞质、核),分子功能条目29个(如蛋白质同源二聚化活性、酶结合)。KEGG通路富集分析得到20条通路。分子对接结果显示,度值排名前3位的活性成分(β-谷甾醇、豆甾醇、金合欢素)与关键抑菌靶点编码蛋白的结合能均<0kJ·mol^(-1)。抑菌试验验证表明,豆甾醇是板蓝根的关键药效成分,其能够下调胞内CASP3和PTGS2蛋白的表达。板蓝根主要通过药效成分豆甾醇作用于PTGS2、CASP3靶点,通过TNF、癌症途径信号通路发挥抑菌作用。

The main antibacterial active ingredients and mechanism of Isatis indigotica were predicted based on network pharmacology. The active ingredients and targets of Isatis indigotica were screened by using TCMSP database;The related targets were searched by OMIM and GeneCards databases with the keyword of “antibacterial”;The intersection target of Isatis indigotica and bacteriostasis were collected through the Venny platform;STRING database combined with Cytoscape software were used to construct the PPI network and screen the key targets;GO function enrichment analysis and KEGG pathway enrichment analysis were carried out by using DAVID database;The key pharmacodynamic ingredients with the antibacterial targets were connected through Autodock Tools 1.5.7 software;The bacteriostatic effects against Escherichia coli(CVCC1515) and targets of the key ingredients were verified by colony counting and SDS-PAGE. A total of 33 active components of Isatis indigotica, 61 potential drug targets, 2 192 bacteriostatic targets, and 32 intersection targets of Isatis indigotica and bacteriostatic action were screened. Protein interaction network analysis revealed that 30 proteins, including TNF, TP53, CASP3, JUN, ESR1 and PTGS2 and so on, might be the key targets of antibacterial activity of Isatis indigotica. The GO functional enrichment analysis yielded 126 entries, including 86 biological process items(e.g. response to estradiol apoptotic signaling pathway, positive regulation of neuron apoptotic process), 11 cell composition items(e.g. cytoplasm, nucleus), and 29 molecular function items(e.g protein homodimerization activity, enzyme binding). KEGG pathway enrichment analysis yielded 20 related signal pathways. Molecular docking result showed that the binding energies of the key active components(beta-sitosterol, stigmasterol and acacetin) with the coding proteins of key antibacterial targets were all less than 0 kJ·mol^(-1). Antibacterial experiments showed that stigmasterol was the key active component of Isatis indigotica which might down-regulated the expression of intracellular CASP3 and PTGS2 proteins. Stigmasterol of the active components of Isatis indigotica exerts antibacterial effect through the targets of CASP3 and PTGS2 which involved in TNF signaling pathway and pathways in cancer.