基于生物信息学和细胞实验验证探讨甘草干姜汤治疗哮喘的作用机制

Effects of Gancao Ganjiang Decoction(甘草干姜汤)on Asthma Based on Bioinformatics and Cellular Experimental Verification

目的:利用网络药理学及分子对接探究甘草干姜汤缓解哮喘症状的潜在作用机制。方法:通过TCMSP数据库及SwissTargetPrediction收集甘草干姜汤的有效成分并进行药物成分靶点的检索;以哮喘为关键词检索GeneCards、OMIM数据库以收集疾病靶点,并将结果与药物成分靶点取交集,交集靶点作为该复方治疗哮喘的潜在作用靶点;对潜在靶点进行蛋白互作关系网络分析,并得到关键靶点。利用DAVID数据库,得到GO和KEGG通路富集分析结果。借助Autodock Vina软件将甘草干姜汤有效成分和蛋白受体进行分子对接模拟。实时荧光定量PCR和ELISA实验分别从mRNA和蛋白层面对甘草干姜汤治疗哮喘的核心靶点及潜在作用机制进行验证。结果:通过网络药理学分析共筛选出甘草干姜汤有效成分97种,哮喘靶点3223个,药物靶点216个,甘草干姜汤治疗哮喘的潜在靶点141个,GO和KEGG富集结果主要为炎症和免疫等相关生物学过程及通路;通过分子对接模拟,验证了甘草干姜汤小分子配体与受体结合能均≤-5.0kcal/mol,结合力良好。PCR结果显示甘草干姜汤可以降低IL-6 mRNA、IL-1βmRNA、TNF-αmRNA及核心靶点RELA mRNA、PTGS2 mRNA、MAP1 mRNA的相对表达量(P<0.05),ELISA结果表明甘草干姜汤可以降低炎症因子IL-6蛋白相对表达量。结论:甘草干姜汤可能是通过抑制气道炎症及调控MAPK和NF-κB信号通路上关键靶点的表达而缓解哮喘。

Objective:To investigate the potential mechanisms of Gancao Ganjiang decoction in alleviating asthma symptoms by network pharmacology and molecular docking.Methods:Firstly,the active ingredients and action targets of Gancao Ganjiang decoction were collected through TCMSP database and Swiss Target Prediction.The related targets were searched with the help of GeneCards and OMIM databases with asthma as the keyword,and the results were intersected with the active ingredient targets.The intersection targets are taken as the potential targets for treating asthma.Secondly,the protein-protein interaction network analysis is carried out on the potential targets to acquire the key targets of Gancao Ganjiang decoction in the treatment of asthma.The enrichment analysis results of GO and KEGG pathways were obtained by using the DAVID database.Molecular docking simulation of the active ingredients and protein receptors of Gancao Ganjiang decoction were carried out with the help of Autodock Vina software.Quantitative Real-time PCR and ELISA experiments verified the core targets and potential mechanisms of Gancao Ganjiang decoction in the treatment of asthma from the mRNA and protein levels,respectively.Results:A total of 97 active ingredients of Gancao Ganjiang decoction,3223 asthma targets,216 drug targets,and 141 potential targets of Gancao Ganjiang decoction in treating asthma were screened out through network pharmacology analysis.GO and KEGG enrichment results showed that the treatment mechanisms of drug were related to biological processes and pathways about inflammation and immunity.Molecular docking verified that the affinity energy of molecule ligands and receptors of Gancao Ganjiang decoction was less than or equal to-5 kcal/mol,and the binding force was stable.The PCR results showed that Gancao Ganjiang decoction could reduce IL-6 mRNA,IL-1βmRNA and TNF-αmRNA relative expression levels,as well as the core targets RELA mRNA,PTGS2 mRNA and MAP1 mRNA(P<0.05).ELISA results showed that Gancao Ganjiang decoction could reduce the relative expression level of inflammatory factor IL-6 protein.Conclusion:Gancao Ganjiang decoction may relieve asthma by inhibiting airway inflammation and regulating the expression of key targets in MAPK and NF-κB signaling pathways.