基于代谢组学的桔梗皂苷D镇咳祛痰活性及作用机制阐释研究

Study on the antitussive and expectorant activities and mechanism of platycodin D based on metabolomics method

本文通过构建小鼠浓氨水引咳模型与小鼠气管酚红排泄模型研究桔梗皂苷D(platycodin D,PD)的镇咳祛痰活性,采用代谢组学方法研究并阐释PD的镇咳祛痰机制。动物实验经江西中医药大学动物伦理委员会批准(批准号:JZLLSC-20220739)。将小鼠随机分为正常组、模型组、阳性药、PD低、中、高剂量给药组,通过浓氨水引咳和酚红排泄实验评估PD的镇咳与祛痰药效。采用UHPLC-LTQ-Orbitrap-MS对小鼠肺组织代谢物进行鉴定,应用正交偏最小二乘判别分析(OPLS-DA)分析方法,通过变量投影重要性值(VIP)和t检验结果筛选差异代谢物,利用MetaboAnalyst平台富集差异性代谢产物的通路。采用比较学方法比较PD与桔梗总皂苷及去芹糖桔梗皂苷D(deapio-platycodin D,DPD)的镇咳祛痰机制的异同。结果显示,各浓度PD能显著延长(P<0.05)浓氨水引咳模型中小鼠的潜伏期,减少咳嗽次数,能显著增加(P<0.05)酚红排泄模型中小鼠的酚红排泄量。代谢组学分析结果显示其可调控苯丙氨酸、酪氨酸和色氨酸生物合成、亚油酸代谢、苯丙氨酸代谢、甘油磷脂代谢、酪氨酸代谢等6条代谢通路发挥镇咳作用;可调控亚油酸代谢、乙醛酸和二羧酸代谢、甘油磷脂代谢、柠檬酸循环、花生四烯酸代谢等8条代谢通路发挥祛痰作用;但受桔梗皂苷成分结构及其与肠道菌群双向调控作用影响,仅亚油酸代谢和花生四烯酸代谢通路均可被桔梗总皂苷、PD及DPD所共同调控而发挥镇咳祛痰作用。生物信息功能分析显示,PD调控的代谢通路与抗炎、免疫功能调节、神经递质释放、细胞信号传导、能量代谢与细胞凋亡等调控机制密切相关。本研究显示PD具有良好的镇咳祛痰活性,PD与桔梗总皂苷及DPD镇咳祛痰机制的异同特性表明PD所含芹糖分子及桔梗皂苷成分与肠道菌群的双向调控作用对于PD及DPD等桔梗皂苷成分的镇咳祛痰机制具有重要影响。

In this paper,the antitussive and expectorant activity of platycodin D(PD)were studied by constructing a mouse cough induced by concentrated ammonia water and a mouse trachea phenol red excretion model.The mechanism of antitussive and expectorant effect of PD was studied by metabolomics.The animal experiment was approved by the Animal Ethics Committee of Jiangxi University of Chinese Medicine(approval number:JZLLSC-20220739).Then mice were randomly divided into the normal,model,positive drug,PD lowdose,PD medium-dose and PD high-dose group.The antitussive and expectorant effects of PD were evaluated using a cough mouse model induced by concentrated ammonia water and a mouse tracheal phenol red excretion model,respectively.UHPLC-LTQ-Orbitrap-MS was used to identify the metabolites of mouse lung tissue,and multivariate statistical analysis method of orthogonal partial least squares discriminant analysis(OPLS-DA)was used for metabolites profile analysis.The differential metabolites were screened by variable projected importance value(VIP)and t-test results.Pathways for enrichment of differentiated metabolites were analyzed using the MetaboAnalyst platform.The comparative method was applied to analyze the differences in mechanisms of PD,Deapio-platycodin D(DPD)and total platycosides fraction.The results showed that PD at different concentrations could significantly prolong(P<0.05)the incubation period of cough mice induced by ammonia water,reduce the coughs frequency,and significantly increase(P<0.05)the amount of phenol red excretion in phenol red excretion model mice.PD could regulate 6 metabolic pathways of phenylalanine,tyrosine and tryptophan biosynthesis,linoleic acid metabolism,phenylalanine metabolism,glycerophospholipid metabolism,and tyrosine metabolism to exert antitussive effect.It could also regulate 8 metabolic pathways of linoleic acid metabolism,glyoxylic acid and dicarboxylic acid metabolism,glycerol phospholipid metabolism,citric acid cycle and arachidonic acid metabolism to exert an expectorant effect.However,only linoleic acid metabolism and glycerophospholipid metabolism could be regulated by the PD,total platycosides fraction and DPD,which may be ascribed to the structural difference of the platycosides and the interaction between platycosides and the intestinal microbiota.Functional analysis showed that these metabolic pathways are closely related to the regulatory mechanisms of anti-inflammatory response,immune function regulation,neurotransmitter release,cell signal transduction,energy metabolism and cell apoptosis.This study shows that PD possesses good antitussive and expectorant activities.In addition,the mechanism difference of PD,total platycosides fraction and DPD imply that the apiose in PD and the interaction between PD and intestinal microbiota could exert an important effect on the antitussive and expectorant mechanism of the platycosides.