基于网络药理学的急支糖浆治疗咳嗽的作用机制研究及实验验证

Mechanism of Jizhi Syrup for treatment of cough based on network pharmacology and experimental verification

目的运用网络药理学的方法挖掘中药复方急支糖浆治疗咳嗽的有效成分、关键靶点以及潜在作用通路。方法分别利用中药系统药理学数据库与分析平台(TCMSP)和Genecards数据库筛选急支糖浆的活性成分作用靶点和咳嗽的疾病靶点,两者靶点取交集后利用Cytoscape软件构建成分-靶点网络,String数据库构建蛋白质相互作用(PPI)网络,拓扑分析获取关键靶点和成分。通过David数据库进行基因本体论(GO)生物学功能富集分析及京都基因和基因组百科全书(KEGG)信号通路富集分析。通过分子对接技术进一步佐证网络药理学结果的可靠性,通过小鼠咳嗽模型进行实验验证。结果急支糖浆中共筛选出活性成分163种,可干预咳嗽相关靶点154个,GO富集586个,KEGG通路119条。其中,槲皮素、山柰酚、β-谷甾醇等可能为急支糖浆治疗咳嗽的关键成分,CXCL8、MAPK8、STAT3、TNF等为潜在靶点,GO富集分析的生物功能涉及到细胞增殖、血管生成、炎症反应等,KEGG通路富集分析涉及到TNF、PI3K-Akt、FoxO、HIF-1等信号通路。超过90%的分子对接结果均具有一定的结合活性。使用流式细胞术、实时荧光定量PCR(qRT-PCR)、酶联免疫吸附(ELISA)的方法验证了急支糖浆对咳嗽小鼠淋巴细胞分化、炎症因子和部分关键靶点表达的影响。结论急支糖浆治疗咳嗽的作用机制是通过多种活性成分作用于多靶点、多通路产生了调控炎症过程、调节免疫功能、促进细胞增殖、修复受损机体等综合效果,该结果为进一步研究急支糖浆有效成分和作用机制奠定基础。

Objective To explore effective ingredients,key targets,and potential pathways of traditional Chinese medicine compound Jizhi Syrup for treatment of cough by method of network pharmacology.Methods TCMSP and genecards databases were used to screen active component action targets of Jizhi Syrup and disease targets of cough.After intersection of two targets,the component target network was constructed by Cytoscape software,and the protein-protein interaction network(PPI)was constructed by string database.The key targets and components were obtained by topological analysis.Gene Ontology(GO)biological function enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)signal pathway enrichment analysis were carried out through David database.The reliability of network pharmacological results was further confirmed by molecular docking technology,and verified by mouse cough model.Results A total of 163 active ingredients were selected from Jizhi Syrup,which could intervene 154 cough related targets,586 GO enrichment and 119 KEGG pathways.Quercetin,kaempferolβ-sitosterol may be the key components of Jizhi Syrup in treatment of cough.CXCL8,MAPK8,STAT3 and TNF are potential targets.The biological functions of GO enrichment analysis involve cell proliferation,angiogenesis and inflammatory response.KEGG pathway enrichment analysis involves TNF,PI3K-Akt,FOXO,HIF-1 and other signal pathways.More than 90%of the molecular docking results have certain binding activity.The effects of Jizhi Syrup on lymphocyte differentiation,inflammatory factors and the expression of some key targets in cough mice were verified by flow cytometry,real-time fluorescence quantitative PCR(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA).Conclusion The mechanism of Jizhi Syrup in treatment of cough is that multiple active components act on multiple targets and pathways,resulting in comprehensive effects of regulating inflammatory process,regulating immune function,promoting cell proliferation and repairing damaged body.The results lay a foundation for the further study of the effective components and mechanism of Jizhi Syrup.