基于UPLC-Q-Orbitrap-MS和网络药理学的覆盆子治疗2型糖尿病作用机制研究

Mechanismresearch of Rubus chingii Hu for T2DM based on UPLC-Q-Orbitrap-MS and network pharmacology

目的 采用超高效液相色谱串联四级杆-静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap-MS)分析覆盆子化学成分,并结合网络药理学和分子对接方法预测其治疗2型糖尿病的有效成分和靶点,探讨其潜在的作用机制。方法 采用Waters CORTECS C18色谱柱,以0.1%甲酸水-甲醇为流动相进行梯度洗脱,全扫描/数据依赖二级质谱扫描(Full MS-dd MS2)模式分析,根据碎片信息,通过参考文献和对照品比对方法对覆盆子成分进行鉴定;通过中药系统药理学数据库与分析平台(TCMSP)对覆盆子活性成分及靶点进行筛选及补充,采用Gene Cards、OMIM和Drug Bank数据库检索2型糖尿病相关靶点,合并取交集,结合STRING数据库构建蛋白相互作用(PPI)网络,应用Cytoscape软件实现“成分-疾病-靶点”网络的可视化,并通过DAVID数据库进行基因本体论(GO)与京都基因与基因组百科全书(KEGG)通路富集富集分析,预测其可能的作用机制;采用分子对接法验证覆盆子主要活性成分与核心靶点的相互作用。结果 共鉴定出了36种化合物,通过数据库筛选8个活性成分和103个“药物-疾病”共同靶点,主要活性成分有槲皮素、山奈酚、甾谷醇、鞣花酸等,关键靶点主要涉及Akt1、IL6、TP53、TNF、VEGFA和MAPK1等。KEGG通路分析主要涉及癌症途径、糖尿病并发症中的AGE-RAGE信号通路、TNF信号通路和HIF-1信号通路等相关通路。分子对接结果显示槲皮素、山奈酚与TP53的亲和力最强,鞣花酸、儿茶素与TNF的结合能力最强,甾谷醇与VEGFA的结合能力最强。结论 覆盆子中多种成分通过作用于多个靶点、多条通路共同发挥降血糖的作用,为探索覆盆子药效物质基础提供了新的思路,为覆盆子的进一步开发利用提供理论依据。

Objective To study and analyze the main chemical constituents of Rubus chingii Hu by UPLC-Q-Orbitrap-MS and predict the effective substances for Type 2 diabetes(T2 DM) according network pharmacology and molecular docking,to explore the potential mechanism.Methods Using Waters CORTECS C18 chromatographic column,gradient elution with 0.1% formic acid water-methanol as mobile phase,Full MS-ddMS2 mode analysis.According to the fragment information,the components of Rubus chingii Hu are identified through the comparison of references and reference substances.The active components and related targets of Rubus chingii Hu were screened and supplemented by the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),the influenza-related targets were searched by GeneCards、OMIM and DrugBank database.Then get the intersection targets of Rubus chingii Hu and T2 DM.The protein-protein interaction(PPI) network was constructed with STRING database,and the visualization of "component-disease-target" network was realized by Cytoscape software.Gene ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were carried out by DAVID database to predict its potential mechanism.Results36 chemical constituents of Rubus chingii Hu were analyzed.A total of 8 active ingredients and 103 "drugdisease" common targets were supplemented through database screening.The main active ingredients included quercetin,kaempferol,sitosterol,and ellagic acid,etc.The key targets involved AKT1、IL6、TP53、TNF、VEGFA and MAPK1,etc.The result of KEGG pathway analysis showed that the treatment of T2 DM by Rubus chingii Hu mainly included cancer pathways,AGE-RAGE signaling pathway in diabetic complications,TNF signaling pathway,HIF-1 signaling pathway.The results of molecular docking showed that quercetin,kaempferol had strong affinity with TP53.Catechin,ellagic acid had the strongest binding ability with TNF.sitosterol had the strongest binding ability with VEGFA.Conclusion The various components of Rubus chingii Hu act on multiple targets and pathways to treat T2 DM,which provides new ideas for exploring the material basis medicinal effects and provides some theoretical basis for the further development and utilization of Rubus chingii Hu.