UPLC-Q-TOF-MS结合网络药理学及实验验证探讨穴位贴敷药物成分及其干预支气管哮喘的作用机制

Components of drugs in acupoint sticking therapy and its mechanism of intervention on bronchial asthma based on UPLC-Q-TOF-MS combined with network pharmacology and experimental verification

运用UPLC-Q-TOF-MS、网络药理学和实验验证探讨穴位贴敷(acupoint sticking therapy,AST)干预支气管哮喘(bronchial asthma,BA)的作用机制。TCMSP数据库中检索白芥子、延胡索、甘遂、细辛和生姜的化学成分作为自建库,采用UPLC-Q-TOF-MS解析AST中的活性成分,在TCMSP和SwissTargetPrediction中筛选靶点;在GeneCards中收集BA靶点,运用Venny 2.1.0平台,对活性成分靶点与BA靶点取交集获得潜在靶点;将潜在靶点导入STRING和DAVID中进行PPI、GO和KEGG分析;建立屋尘螨(house dust mite,HDM)诱导的哮喘小鼠,通过肺功能、免疫印迹法(Western blot)和流式细胞术(flow cytometry)等研究AST对哮喘小鼠的作用机制。结果显示,UPLC-Q-TOF-MS得到54个活性成分,交集得到162个潜在靶点,选取前53个作为关键靶点。PPI分析、GO和KEGG分析发现,AST可能通过清风藤碱、芥子酸、二氢辣椒素、6-姜酚等活性成分,作用于SRC、PIK3CA等靶点,调节PI3K-AKT、ErbB、趋化因子、sphingolipid等信号通路以干预BA病理机制。AST能改善哮喘小鼠肺功能,下调肺组织中PI3K和p-AKT蛋白表达,增强PETN蛋白表达以及降低肺组织中Ⅱ型固有免疫细胞(typeⅡinnate lymphoid cells,ILC2s)水平。综上可得,AST可能主要通过下调PI3K-AKT通路,抑制ILC2s,从而减轻哮喘气道炎症、降低气道高反应等。

UPLC-Q-TOF-MS combined with network pharmacology and experimental verification was used to explore the mechanism of acupoint sticking therapy(AST)in the intervention of bronchial asthma(BA).The chemical components of Sinapis Semen,Cory-dalis Rhizoma,Kansui Radix,Asari Radix et Rhizoma,and Zingiberis Rhizoma Recens were retrieved from TCMSP as self-built database.The active components in AST drugs were analyzed by UPLC-Q-TOF-MS,and the targets were screened out in TCMSP and Swiss-TargetPrediction.Targets of BA were collected from GeneCards,and the intersection of active components and targets was obtained by Venny 2.1.0.The potential targets were imported into STRING and DAVID for PPI,GO,and KEGG analyses.The asthma model induced by house dust mite(HDM)was established in mice.The mechanism of AST on asthmatic mice was explored by pulmonary function,Western blot,and flow cytometry.The results indicated that 54 active components were obtained by UPLC-Q-TOF-MS and 162 potential targets were obtained from the intersection.The first 53 targets were selected as key targets.PPI,GO,and KEGG analyses showed that AST presumedly acted on SRC,PIK3 CA,and other targets through active components such as sinoacutine,sinapic acid,dihydrocapsaicin,and 6-gingerol and regulated PI3 K-AKT,ErbB,chemokine,sphingolipid,and other signaling pathways to intervene in the pathological mechanism of BA.AST can improve lung function,down-regulate the expression of PI3 K and p-AKT proteins in lung tissues,enhance the expression of PETN protein,and reduce the level of typeⅡinnate immune cells(ILC2 s)in lung tissues of asthmatic mice.In conclusion,AST may inhibit ILC2 s by down-regulating the PI3 K-AKT pathway to relieve asthmatic airway inflammation and reduce airway hyperresponsiveness.