基于HPLC-Q/TOF MS与网络药理学结合分子对接技术探讨清肺汤入血成分的镇咳作用机制

Analysis of the antitussive mechanism of blood compounds of Qingfei Decoction based on the technology of HPLC-Q/TOF MS and network pharmacology with molecular docking

目的建立基于HPLC-Q/TOF MS的方法鉴定清肺汤的活性成分,并基于网络药理学的方法寻找清肺汤镇咳的潜在活性机制。方法将10只Wistar大鼠连续灌胃7 d给予清肺汤水煎液后,收集2 h的血浆。样品前处理后,用ACQUITY HPLC HSS Column(100 mm×3.0 mm,1.8μm)色谱柱以质量分数为0.2%甲酸水溶液(A)-乙腈(B)为流动相,梯度洗脱。在电喷雾离子源(ESI源),正负离子模式下分析入血成分;通过SwissTargetPrediction数据库筛选入血成分的潜在靶标,与在GeneCards数据库、CTD数据库和Open Targets Platform数据库中筛选得到的咳嗽靶标匹配获取交集靶标;通过Metascape数据库进行GO生物学分析和KEGG通路分析;通过STRING数据库与Cytoscape软件绘制PPI网络,通过节点和边缘集群算法寻找核心靶点,并用分子对接进行验证。结果通过入血成分的定性分析,检测出了59个活性成分,99个清肺汤镇咳的潜在靶标。GO分析表明清肺汤发挥镇咳作用主要富集在刺激反应与代谢调控等生物过程,细胞膜、神经突触等细胞组成,结合功能、激酶活性等分子功能。关键通路多与免疫调节、炎症反应以及气道反应和气道重塑有关。筛选出BCL2L1、JUN、STAT3、MMP9、HIF1A等核心靶点。结论清肺汤通过多成分、多靶点、多通路发挥镇咳作用.

Objective To establish a method based on HPLC-Q/TOF MS to identify the active ingredients of Qingfei Decoction,and to find the potential active mechanism of Qingfei Decoction for antitussive based on the method of network pharmacology.Methods 10 Wistar rats were intragastrically administered with Qingfei Decoction for 7 days,and the plasma was collected for 2 hours.The sample was separated on an ACQUITY HPLC HPLC HSS column(100 mm×3.0 mm,1.8μm)with a gradient elution consisted of 0.2%formic acid water(A)-acetonitrile(B)as mobile phase.The plasma components were analyzed in positive and negative ion mode by Electrospray ion source(ESI source).The potential targets of blood components were screened through the SwissTargetPrediction database,and the targets of cough were screened in the GeneCards database,CTD database and Open Targets Platform database.The intersection targets of both were regarded as targets which played a role of cough relieving in Qingfei Decoction.GO biological analysis and KEGG pathways analysis were conducted through Metascape database.The PPI network was drawn through STRING database and Cytoscape software.The key targets were selected by node and edge clustering algorithm,and molecular docking was used for verification.Results Through the qualitative analysis of blood components,59 active ingredients and 99 potential targets of Qingfei Decoction for antitussive were detected.GO analysis showed that the antitussive effect of Qingfei Decoction was mainly concentrated in biological processes such as stimulation response and metabolic regulation,cell components such as cell membrane and nerve synapse,molecular functions such as binding function and kinase activity.The pathways were mostly related to immune regulation,inflammatory response,airway response and airway remodeling.Key targets such as BCL2L1,JUN,STAT3,MMP9,and HIF1A were screened out.Conclusion Qingfei Decoction exerts antitussive effects through multiple components,multiple targets,and multiple pathways.