基于LC-MS/MS和靶点网络探讨扶正透邪解毒化瘀方对多重耐药铜绿假单胞菌的干预机制

Study on the intervention mechanism against multidrug-resistant Pseudomonas aeruginosa by Fuzheng Touxie Jiedu Huayu Formula based on LC-MS/MS and target network

关键词:  扶正透邪解毒化瘀方  铜绿假单胞菌  多重耐药  药物靶点网络  联合用药  抑菌

Key words:  Fuzheng Touxie Jiedu Huayu Formula  Pseudomonas aeruginosa  multidrug-resistant  drug targets network  combination of drugs  antibacterial

摘要:

[目的] 从药物作用靶点层面探讨扶正透邪解毒化瘀方体外干预多重耐药铜绿假单胞菌的药效物质基础和直接/协同机制。[方法] 通过液相色谱-串联质谱联用（LC-MS/MS）、中药系统药理数据库和分析平台（TCMSP）筛选扶正透邪解毒化瘀方化学成分；通过Stitch数据库查找上述成分以及亚胺培南、头孢他啶、环丙沙星作用于铜绿假单胞菌的蛋白靶点；运用Cytoscape 3.9.1构建“中药—化学成分—作用靶点网络图”和“蛋白质-蛋白质互作网络”；筛选该方与抗菌药物共同作用靶点；借助DAVID数据库对靶点进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。[结果] LC-MS/MS共鉴定出108个化学成分，TCMSP数据库共筛选出155个化学成分，去重后共得到162个化学成分，其中42个化学成分能够直接作用于铜绿假单胞菌240个靶蛋白；网络药理学筛选出17个核心成分，以黄酮类、甾醇类、萜类为主，其中β-谷甾醇、槲皮素、木犀草素度值较高；该方分别与亚胺培南、头孢他啶、环丙沙星有3/3/17个共同作用靶点，4种药物共同作用靶点2个，共同作用靶点主要作用于细菌能量和生化代谢、DNA复制、蛋白质功能、表面多糖形成等途径；GO和KEGG富集分析提示该方主要与调节细菌能量和生化代谢、调控DNA/蛋白质稳态、影响细菌胞质胞膜以及干预对细菌起保护作用的表面多糖和生物被膜合成等密切相关。[结论] 扶正透邪解毒化瘀方具有多成分、多靶点、多途径干预铜绿假单胞菌的特点，发挥主要干预作用的化学成分以黄酮类、甾醇类、萜类为主，直接/协同抗菌药物干预铜绿假单胞菌的机制与影响细菌遗传、代谢、生物被膜形成等相关。研究从药物作用靶点层面为从天然药物中开发抗菌药物提供了一定的思路和借鉴。

Abstract:

[Objective] To investigate the pharmacodynamic material basis and direct/synergistic mechanism of Fuzheng Touxie Jiedu Huayu Formula in vitro intervention against multidrug-resistant Pseudomonas aeruginosa at the drug targets level. [Methods] The chemical components of Fuzheng Touxie Jiedu Huayu Formula were screened by LC-MS/MS and TCMSP database. The protein targets of the above components as well as imipenem，ceftazidime and ciprofloxacin acting on Pseudomonas aeruginosa were identified by Stitch database. Cytoscape 3.9.1 software was used to construct “herbal-chemical composition-target network” and “protein-protein interaction network”. Venn diagrams were used to screen the formula for co-acting targets with antibacterial drugs. GO functional enrichment analysis and KEGG pathway enrichment analysis of the targets were performed with the aid of the DAVID database. [Results] A total of 108 chemical components were identified by LC-MS/MS，155 chemical components were screened from the TCMSP database，and 162 chemical components were obtained after de-duplication，of which 42 chemical components were able to act directly on 240 target proteins of Pseudomonas aeruginosa. 17 core components were screened by network pharmacology，mainly flavonoids，sterols，and terpenoids，among which β-sitosterol，quercetin，and luteolin have higher degree. This formula has 3/3/17 common targets with imipenem，ceftazidime，and ciprofloxacin，and 2 common targets for the 4 drugs. The common targets of actions are mainly in the pathways of bacterial energy and biochemical metabolism，DNA and genetic material replication，protein function，and surface polysaccharide formation. GO and KEGG enrichment analyses suggested that this formula is mainly related to the regulation of bacterial energy and biochemical metabolism，the regulation of DNA/protein homeostasis，the influence on the bacterial cytoplasmic membrane，and the intervention in the synthesis of surface polysaccharides and biofilms，which play a protective role for bacteria. [Conclusion] Fuzheng Touxie Jiedu Huayu Formula has multi-component，multi-target，and multi-channel interventions with Pseudomonas aeruginosa，and the main intervening chemical components are mainly flavonoids，sterols，and terpenoids. The mechanism of direct/synergistic antibacterial drug interventions on Pseudomonas aeruginosa is related to the influence of bacterial genetics，metabolism，biofilm formation，etc. This study provided some ideas and references for the development of antibacterial drugs from natural drugs at the drug targets level.