基于UHPLC-MS的吴茱萸汤影响虚寒呕吐大鼠尿液代谢谱的研究

Effect of Wuzhuyu Decoction on urine metabolic spectrum in rats with deficiency cold and vomit based on UHPLC-MS

目的 观察吴茱萸汤对虚寒呕吐大鼠尿液代谢谱的变化,探讨其治疗虚寒呕吐证可能的作用机制。方法 运用复合法(大黄+顺铂)制备大鼠虚寒呕吐模型,将大鼠随机分为对照组、模型组、吴茱萸汤组。利用超高液相色谱-质谱(UPLC-MS/MS)技术结合主成分分析、偏最小二乘分析等方法对尿液数据进行分析,鉴定出潜在生物标志物,通过VIP及非参数检验筛选并通过多元化ROC曲线验证差异代谢物;运用PathwayAnalysis数据库对差异性代谢物进行拓扑分析;运用R和Cytoscape进行代谢产物的相关性分析和模块化分析。结果 尿液代谢谱对照组和模型组完全分离,吴茱萸汤组靠近对照组,表明模型复制成功,且吴茱萸汤能够干预大鼠的虚寒呕吐症状,提示大鼠机体具有恢复到正常状态的趋势。从尿液中筛选出34种差异代谢物进行通路分析,发现虚寒呕吐发挥干预作用可能与缬氨酸、亮氨酸和异亮氨酸的生物合成,苯丙氨酸、酪氨酸和色氨酸生物合成,精氨酸和脯氨酸代谢,色氨酸代谢,酪氨酸代谢,花生四烯酸代谢,三羧酸循环(TCA循环)等通路有关;进行模块化分析发现10个模块之间关系密切,丙氨酸、亮氨酸、酪氨酸、色氨酸、异亮氨酸、琥珀酸、丙氨酸、富马酸、苹果酸、异柠檬酸等生物靶点可作为虚寒呕吐模型的标识。结论 吴茱萸汤能够改善虚寒呕吐模型的生理特征,机制可能与吴茱萸汤调节大鼠体内紊乱的氨基酸代谢、能量代谢及脂代谢等有关。

Objective To observe the changes of urine metabolic profile of Wuzhuyu Decoction in rats with deficiency cold and vomit,and explore its possible mechanism of treatment of deficiency cold and vomit syndrome.Methods A rat model of deficiency cold and vomit was prepared by a composite method(rhucax+cisplatin).Rats were randomly divided into control group,model group,and Wuzhuyu Decoction group.UPLC-MS/MS combined with principal component analysis and partial least squares analysis were used to analyze urine data and identify potential biomarkers.Diversified ROC curves were used to validate differential metabolites;Pathway Analysis database was used for topological analysis of differential metabolites;R and Cytoscape were used for correlation analysis and modular analysis of metabolites.Results The urine metabolic spectrum of control group and model group were completely separated.Wuzhuyu Decoction group was close to the control group,indicating that the model was successfully replicated;And Wuzhuyu Decoction can interfere with the symptoms of deficiency cold and vomit in rats,suggesting that the rat body had a tendency to return to normal state.Through modular analysis of 34 urine differential metabolites,the deficiency cold and vomit treatment was revealed to affect biosynthesis of valine,leucine and isoleucine,phenylalanine,tyrosine and tryptophan biosynthesis,arginine and proline metabolism,tryptophan metabolism,tyrosine metabolism,arachidonic acid metabolism,citric acid cycle(TCA cycle)pathways.Modularity analysis revealed that there was close relationship between 10 modules;Alanine,leucine,tyrosine,tryptophan,isoleucine,succinic acid,alanine,fumaric acid,malic acid,isocitrate,and other biological targets can thus be used as markers of deficiency cold and vomit.Conclusion Wuzhuyu Decoction can improve the physiological characteristics of the model of deficiency cold and vomit.The mechanism may be related to the regulation of amino acid metabolism,energy metabolism and lipid metabolism in rats.