基于网络药理学的双氢杨梅树皮素抗金黄色葡萄球菌活性和作用机制研究

Exploring the Activity and Mechanism of Ampelopsin against Staphylococcus aureus Based on Network Pharmacology

目的:对比双氢杨梅树皮素(Ampelopsin,APS)与四种β-内酰胺类抗生素对不同金黄色葡萄球菌(Staphylococcus aureus,SA)抗菌活性的差异,并通过网络药理学方法推测APS的抗菌作用机制。方法:采用连续稀释法测定APS及β-内酰胺类抗生素对受试菌MSSA-4(不产膜敏感菌)、MRSA-6(不产膜耐药菌),MSSA-11(产膜敏感菌)、MRSA-12(产膜耐药菌)的最低抑菌浓度(Minimal inhibitory concentration,MIC);运用一元多因素方差分析,比较耐药性与生物被膜对受试菌MIC的影响;通过PubChem数据库获取APS化合物的结构,采用PharmMapper数据库进行靶点垂钓,筛选后的靶点导入STRING数据库建立PPI网络图并抓取节点信息,建立“化合物-靶点-靶点互作”网络,利用Metascape平台对关键靶点进行GO富集分析及KEGG通路富集分析,预测其抗菌作用机制。结果:APS对MSSA-4、MRSA-6、MSSA-11、MRSA-12的MIC分别为125、125、62.5、62.5μg/mL;方差分析表明,细菌耐药性及生物被膜对APS的抗菌作用影响不大,且对产膜菌有更好的抗菌活性;而四种β-内酰胺类抗生素则易受到细菌耐药性和生物被膜影响,导致受试菌对抗生素的敏感性降低。通过网络药理学获得APS的潜在抗菌作用靶点123个,蛋白互作网络提示ALB、AKT1、MMP9、MAPK1、CASP3、IGF1、MAPK8、HRAS、BCL2L1、ESR1可能是其抗菌的核心靶点,参与的生物功能主要包括细菌反应、细菌黏附调节、蛋白质结构域特异性结合等,主要作用于局灶性黏附、氨基糖和核苷酸糖代谢、药物代谢等通路。结论:细菌耐药性及生物被膜不会影响APS的抗菌作用;APS具有多靶点、多通路影响细菌生物被膜而发挥抗菌作用的特点。

Objective: Comparison of the antibacterial activity of APS with four β-lactam antibiotics against different Staphylococcus aureus(SA) and speculation of the antibacterial mechanism of action of APS by network pharmacological approach. Methods: The MICs of APS and β-lactam antibiotics on MSSA-4(non-membrane producing sensitive bacteria),MRSA-6(non-membrane producing resistant bacteria), MSSA-11(membrane producing sensitive bacteria) and MRSA-12(membrane producing resistant bacteria) were determined by serial dilution method. The effects of drug resistance and biofilm on MIC were compared by one-way multivariate ANOVA. The structure of APS compound was obtained from PubChem database, and target fishing was carried out using PharmMapper database, and the screened targets were imported into STRING database to establish PPI network diagram and capture node information to establish “compound-target-target interaction” network. Metascape platform was used to perform GO enrichment analysis and KEGG pathway enrichment analysis on key targets to predict their antibacterial mechanism of action. Results: The MIC of MSSA-4, MRSA-6, MSSA-11 and MRSA-12 by APS were 125, 125, 62.5 and 62.5 μg/mL, respectively. Analysis of variance showed that bacterial drug resistance and biofilm had little effect on the antibacterial effect of APS, and had better antibacterial activity against membrane producing bacteria. And four β-Lactam antibiotics were easily affected by bacterial drug resistance and biofilm,resulting in the decrease of the sensitivity of the tested bacteria to antibiotics. 123 potential antimicrobial action targets of APS were obtained by network pharmacology, and the protein interaction network had suggested that ALB, AKT1, MMP9,MAPK1, CASP3, IGF1, MAPK8, HRAS, BCL2L1, ESR1 might be its core targets for the biological functions involved mainly include bacterial response, bacterial adhesion regulation, protein domain specific binding and so on, and mainly acted on focal adhesion, amino sugar and nucleotide sugar metabolism, drug metabolism and other pathways. Conclusion:Bacterial resistance and biofilm did not affect the antibacterial effect of APS. APS had the characteristics of multi-target and multi-pathway to affect the bacterial biofilm and exert antibacterial effects.