基于抑菌实验和网络药理学探讨D-柠檬烯、2-茨醇对白色念珠菌的抑制作用

Inhibitory Effects of D-Limonene and 2-Zinol on Candida Albicans Were Studied Based on Antibacterial Experiments and Network Pharmacology

目的:采用抑菌实验研究蛇床子-冰片药对成分中的D-柠檬烯及2-茨醇的体外抗白色念珠菌作用,并运用网络药理学预测D-柠檬烯和2-茨醇治疗念珠菌病的核心靶点和通路。方法:以白色念珠菌为研究对象,K-B纸片扩散法分别测定0.5、1.0、1.5 mg的D-柠檬烯、2-茨醇、制霉菌素的药液抑菌圈直径;采用试管双倍稀释法和棋盘法,测定D-柠檬烯、2-茨醇的最低抑菌浓度(MIC)以及两两联用的MIC,计算出联合抑菌分数(FIC)。通过Pubchem、SwissTargetPrediction数据库预测D-柠檬烯、2-茨醇的有效靶点;通过GeneCards、OMIM数据库检索念珠菌病相关的疾病靶点;运用Venny软件获得两种化学成分和念珠菌病的共同靶点;运用Cytoscape 3.9. 0软件构建“成分-靶点-疾病”网络;利用STRING数据库构建蛋白互作PPI网络;利用R软件进行GO功能及KEGG通路富集分析。结果:D-柠檬烯的MIC为5 mg/mL,2-茨醇的MIC为2.5 mg/mL。D-柠檬烯与2-茨醇联用的FIC指数为0.75,呈相加作用。网络药理学筛选得到两种成分相关作用靶点152个,疾病靶点893个,两者交集靶点为24个;网络拓扑分析得到核心靶点为肿瘤坏死因子(TNF)、过氧化物酶体增殖物激活受体γ(PPARG)、雌激素受体(ESR1)等;KEGG分析得到核心通路为C型凝集素受体信号通路(C-type lectin receptor signaling pathway)、Fc epsilon RI信号通路(Fc epsilon RI signaling pathway)、催乳素信号通路(prolactin signaling pathway)等。结论:D-柠檬烯、2-茨醇对白色念珠菌均有抑制作用,且2种组分药物联合使用具有一定的协同作用。网络药理学预测初步提示D-柠檬烯、2-茨醇可能通过作用于TNF、PPARG、ESR1等核心靶点调控C型凝集素受体信号通路(C-type lectin receptor signaling pathway)、Fc epsilon RI信号通路(Fc epsilon RI signaling pathway)等以治疗念珠菌病。

Objective:Bacteriostasis experiment was conducted to study in vitro anti-candida albicans effect of D-limonene and 2-Zinol of couplet medicinals of Fructus cnidii and Borneolum Syntheticum,and network pharmacology was utilized to predict the core targets and pathways of D-limonene and 2-Zinol in the treatment of candidiasis.Methods:Taking Candida albicans as the research object,K-B disk diffusion method was used to determine the diameter of the antibacterial zone of 0.5,1.0,and 1.5 mg of D-limonene,2-Zinol,and nystatin in the drug solution,respectively.The minimum inhibitory concentration(MIC)and the combined MIC of D-limonene and 2-Zinol were determined by tube double dilution method and checkerboard method,and the combined inhibitory fraction(FIC)was calculated.The effective targets of Dlimonene and 2-Zinol were predicted by Pubchem and SwissTargetPrediction database.GeneCards and OMIM databases were used to search the disease targets associated with candidiasis.Venny software was used to obtain the common target of the two chemical components and candidiasis,and the"Component-Targetdisease"network was constructed using Cytoscape 3.9.0 software.Protein interaction PPI network was constructed using STRING database,and GO function and KEGG path enrichment were analyzed using R software.Results:The MIC of D-limonene was 5 mg/mL,and the MIC of 2-Zinol was 2.5 mg/mL.The FIC index of D-limonene combined with 2-Zinol was 0.75,showing additive effect.Network pharmacology screening identified 152 targets related to the two components,893 disease targets,and 24 intersecting targets.Network topology analysis showed that the core targets included tumor necrosis factor(TNF),peroxisome proliferator-activated receptorγ(PPARG)and estrogen receptor(ESR).KEGG analysis showed that the core pathways were C-type lectin receptor signaling pathway and Fc epsilon RI signaling pathway,prolactin signaling pathway and other signaling pathways.Conclusion:Both D-limonene and 2-Zinol have inhibitory effects on Candida albicans,and the combined use of the two components has a certain synergistic effects.Network pharmacological prediction suggests that D-limonene and 2-Zinol may regulate C-type lectin receptor signaling pathway,Fc epsilon RI signaling pathway and other signaling pathways by acting on core targets such as TNF,PPARG and ESR1 to treat candidiasis.