基于网络药理学探讨固表防感加减方在免疫抑制小鼠预防流感病毒中的作用机制及实验验证

Exploring mechanism and experimental validation of Gubiao Fanggan Modified Formula in preventing influenza virus in immunosuppressive mice based on network pharmacology

目的:运用网络药理学方法探讨固表防感加减方调节免疫抑制小鼠防御A型流感病毒的作用靶点及信号通路,并通过免疫抑制小鼠模型进行关键靶点验证。方法:利用TCMSP数据库检索固表防感加减方药物的有效成分,利用GeneCard、OMIM、PharmGkb数据库获得有效成分预防流感的潜在靶点,取其交集靶点;运用STRING11.5数据库制作蛋白质-蛋白质相互作用网络,分析并找到核心靶点;利用Cytoscape3.8.1构建中药-成分-疾病靶点网络,并进行GO富集分析和KEGG富集分析。腹腔注射环磷酰胺构建免疫功能抑制小鼠模型,设置正常组、模型对照组、固表防感加减方组、奥司他韦组,随后预防性给药,第4天进行流感病毒干预,药物灌胃治疗7 d后,通过小鼠脾脏指数、HE染色、RT-qPCR、免疫组化法验证关键靶点。结果:固表防感加减方中5味中药共含82个活性成分,IL-6、TNF-α、IL-2等72个药物与疾病的共同靶点,主要涉及IL-17、TNF及AGE-RAGE信号通路等。固表防感加减方可以提高脾脏指数,显著降低小鼠脾组织中IL-6及TNF-α的mRNA和蛋白表达(P<0.05或P<0.01)。结论:固表防感加减方可能通过IL-6和TNF-α等靶点调控机体免疫功能,从而预防流感病毒感染。

Objective:To invastigate the targets and signaling pathways of Gubiao Fanggan Modified Formula in regulating the defense against influenza A virus in immunosuppressed mice by network pharmacology,and the key targets were verified by immunosuppressive mice model.Methods:TCMSP database was used to search the active ingredients of Gubiao Fanggan Modified Formula,and GeneCard,OMIM,PharmGkb databases were used to obtain the potential targets of the active ingredients to prevent influenza,and take their intersection targets were taken;STRING11.5 database was used to make protein-protein interaction network analyzed and finded the core targets;Cytoscape3.8.1 was used to build a traditional Chinese medicine-ingredient-disease target network,and GO enrichment analysis and KEGG enrichment analysis were performed.Intraperitoneal injection of cyclophosphamide was used to construct a mouse model of immune function suppression,normal group,model control group,Gubiao Fanggan Modified Formula group and oseltamivir group were set up,followed by prophylactic administration,and influenza virus intervention was performed on the fourth day.After 7 days of intragastric administration,the key targets were verified by mouse spleen index,HE staining,RT-qPCR and immunohistochemistry.Results:There were 82 active ingredients in five traditional Chinese medicines in Gubiao Fanggan Modified Formula,and 72 common targets of drugs and diseases such as IL-6,TNF-α,IL-2,etc,mainly involving IL-17,TNF and AGERAGE signaling pathway.Gubiao Fanggan Modified Formula could increase spleen index and significantly reduce mRNA and protein expressions of IL-6 and TNF-αin spleen tissue of mice(P<0.05 or P<0.01).Conclusion:Gubiao Fanggan Modified Formula may regulate body's immune function through targets such as IL-6 and TNF-α,thereby preventing influenza virus infection.