基于中药血清药物化学的桂枝加葛根汤干预流感病毒性肺炎功效组分表征

Characterization of Effective Components in Guizhi Jia Gegentang for Intervention of Influenza Virus Pneumonia Based on Serum Pharmacochemistry of Traditional Chinese Medicine

目的:采用超高效液相色谱-四级杆-静电场轨道阱高分辨质谱法(UPLC-Q-Exactive Orbitrap MS)对桂枝加葛根汤(GGT)干预流感病毒性肺炎的功效组分进行表征。方法:雄性BALB/c小鼠12只,随机分为空白组和GGT组(36 g·kg^(-1)·d^(-1)),每组6只,GGT组连续灌服GGT提取液5d,空白组灌服等量超纯水。给药结束后收集血清及肺组织,应用UPLC-Q-Exactive Orbitrap MS表征小鼠血清及肺组织中GGT原型及代谢成分。同时存在于小鼠给药血清及肺组织中的GGT成分定义为其功效组分,利用SwissTargetPrediction数据库对功效组分的靶点进行检索,通过GeneCards数据库查询流感病毒性肺炎的靶点,进而取交集得到GGT干预疾病的潜在靶点。通过STRING数据库对潜在靶点进行蛋白质-蛋白质相互作用(PPI)网络分析,利用Metascape对潜在靶点进行京都基因与基因组百科全书(KEGG)通路富集分析。结果:在小鼠含药血清中共检测到GGT原型成分29个,代谢成分28个,其中有11个原型成分和4个代谢成分在小鼠肺组织中被检测到。主要代谢途径包括还原、羟基化、甲基化、葡萄糖醛酸化和硫酸化。PPI网络及KEGG通路富集分析结果显示这些功效组分可能通过作用于白蛋白(ALB)、表皮生长因子受体(EGFR)、类固醇受体辅助活化因子(SRC)、Toll样受体4(TLR4)等靶点及核转录因子-κB(NF-κB)及黏附连接等信号通路发挥作用。结论:同时存在于小鼠含药血清及肺组织中的11个原型成分及4个代谢物可能是GGT干预流感病毒性肺炎的潜在功效组分,并通过干预炎症代谢通路发挥作用,可为GGT治疗流感病毒性肺炎的机制研究提供参考。

Objective:To characterize the efficacy components of Guizhi Jia Gegentang(GGT)in intervening influenza virus pneumonia by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry(UPLC-Q-Exactive Orbitrap MS).Method:BALB/c mice were randomly divided into normal group and GGT group(36 g·kg^(-1)·d^(-1))with six mice in each group.GGT group was continuously administered GGT extract for 5 d,while the normal group was administered an equal amount of ultrapure water.Serum and lung tissue were collected after administration,and UPLC-Q-Exactive Orbitrap MS was used to characterize the prototypical and metabolic components of GGT in serum and lung tissue of mice.The components existed simultaneously in the serum and lung tissue of mice from the GGT group were defined as its functional components,and the targets of efficacy components were searched by SwissTargetPrediction database,and GeneCards database was used to query the target of influenza virus pneumonia,and then the intersection was taken to obtain potential targets of GGT for intervening in the disease.Protein-protein interaction(PPI)network analysis of potential targets was performed by STRING database,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis on potential targets was performed by Metascape.Result:A total of 29 prototypical components and 28 metabolic components of GGT were detected in the drug-containing serum of mice,of which 11 prototypical components and 4 metabolic components were detected in the lung tissue of mice.The main metabolic pathways included reduction,hydroxylation,methylation,glucuronidation and sulfation.The results of PPI network and KEGG analysis showed that these functional components may act through their effects on targets such as albumin(ALB),epidermal growth factor receptor(EGFR),steroid receptor coactivator(SRC),Toll-like receptor 4(TLR4),nuclear transcription factor(NF)-κB and adhesion junction.Conclusion:The 11 prototypical components and 4 metabolites present simultaneously in the drug-containing serum and lung tissue of mice may be the potential therapeutic components of GGT in interfering with influenza viral pneumonia,and act through interfering with inflammatory metabolic pathways.This study can provide a reference for the mechanism study of GGT in the treatment of influenza viral pneumonia.