基于网络药理学和动物体内试验验证云实感冒合剂对呼吸道合胞病毒作用  

Verification of Yunshi Ganmao Heji efficacy against respiratory syncytial virus based on network pharmacology and animal experiments

作　　者：徐庆胜 李玉迁 蒋立娜 魏洪 迟茜文 廖思睿 陈维娇 Xu Qingsheng;Li Yuqian;Jiang Lina;Wei Hong;Chi Qianwen;Liao Sirui;Chen Weijiao(National Experimental Teaching Center,Department of Microbiology,School of Basic Medical Sciences,Guizhou Provincial Key Laboratory of Pathogen Biology,Guizhou Medical University,Guiyang 550025,China;Department of Microbiology,School of Basic Medical Sciences,Guizhou Provincial Key Laboratory of Pathogen Biology,Guizhou Medical University,Guiyang 550025,China;National Experimental Teaching Center,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院国家级实验教学中心,微生物学教研室,贵州省普通高校学校病原生物学特色重点实验室,贵阳550025 [2]贵州医科大学基础医学院微生物学教研室,贵州省普通高校学校病原生物学特色重点实验室,贵阳550025 [3]贵州医科大学基础医学院国家级实验教学中心,贵阳550025

出　　处：《中华微生物学和免疫学杂志》2024年第2期170-182,共13页Chinese Journal of Microbiology and Immunology

基　　金：贵州省中医药、民族医药科学技术研究专项(202449782);2022年省级大学生创新训练项目(S202210660133);2023年省级大学生创新训练项目(S202310660027、S202310660049);国家科技部及教育部"111计划"(D20009)。

摘　　要：目的基于网络药理学和体内试验探讨云实感冒合剂抗呼吸道合胞病毒(respiratory syncytial virus,RSV)的作用机制。方法网络药理学预测:运用TCMSP、GeneCards等数据库预测云实感冒合剂干预RSV的活性成分及作用靶点。利用Cytoscape 3.2.1软件构建中药成分-疾病靶点网络图。通过STRING数据库分析蛋白质相互作用关系。借助Metascape数据库进行富集分析。采用分子对接技术分析验证网络药理学结果。云实感冒合剂干预RSV感染验证试验:滴鼻法建立RSV小鼠模型,灌胃合剂后检测血常规、肺指数,观察肺组织病理变化,流式细胞术检测外周血T细胞亚群,ELISA检测血清TNF-α、IL-6、IL-1β含量,RT-PCR检测肺组织中TLR4、NF-κB和RSV-N基因mRNA相对水平。结果分析获得云实感冒合剂有效活性成分41个,抗RSV靶点111个,富集得到167条信号通路,主要是PI3K/AKT、MAPK、Toll-like等信号通路。分子对接结果显示,云实感冒合剂活性成分木犀草素、山奈酚、槲皮素与RSV-G、RSV-F、PI3K、AKT1、Bcl-2结合能均小于0 kcal/mol。体内试验结果显示,与RSV组相比,云实感冒合剂高剂量组白细胞、淋巴细胞计数升高、肺指数降低,差异有统计学意义(P<0.05)。HE染色显示RSV组肺组织增生,肺泡壁变厚,间质内有炎性细胞浸润,利巴韦林组和云实感冒合剂低、中、高剂量组中肺泡大小趋向均匀,肺泡壁厚度均一。与RSV组相比,云实感冒合剂低、中、高剂量组的CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞数量明显升高,CD4^(+)/CD8^(+)T细胞数量比值降低,血清中TNF-α、IL-6、IL-1β含量下降,肺组织中病毒载量和TLR4、NF-κB mRNA水平降低,差异均具有统计学意义(P<0.05)。结论云实感冒合剂通过多成分、多靶点、多通路发挥调控RSV感染,主要通过缓解肺组织病理损伤,减轻炎症反应,其机制可能与抑制TLR4/NF-κB通路激活有关。Objective To investigate the mechanism of Yunshi Ganmao Heji against respiratory syncytial virus(RSV)infection based on network pharmacology and in vivo experiments.Methods Network pharmacological prediction:Several databases including TCMSP and GeneCards were used to predict the active ingredients and targets of Yunshi Ganmao Heji in the intervention of RSV infection.Cytoscape 3.2.1 software was used to construct the traditional Chinese medicine component-disease target network diagram.The interactions between proteins were analyzed by STRING database.GO functional enrichment analysis and KEGG pathway enrichment analysis were performed using Metascape database.Molecular docking technology was used to verify the results of network pharmacology.Experimental verification of Yunshi Ganmao Heji for the intervention of RSV infection:A mouse model of RSV infection was established through intranasal infection.After the administration of Yunshi Ganmao Heji,blood routine test results,lung indexes and pathological changes in lung tissue were analyzed.Peripheral blood T cell subsets were detected by flow cytometry.The levels of TNF-α,IL-6 and IL-1βin serum were detected by ELISA.RT-PCR was used to detect the relative expression of TLR4,NF-κB and RSV-N gene at mRNA level in lung tissues.Results A total of 41 active ingredients of Yunshi Ganmao Heji and 111 drug targets for RSV infection were obtained.Besides,167 signaling pathways mainly including PI3K/AKT,MAPK and Toll-like receptor signaling pathways were obtained.Molecular docking results showed that the binding energies of luteotin,kaempferol and quercetin,three active ingredients of Yunshi Ganmao Heji,with RSV-G,RSV-F,PI3K,AKT1 and Bcl-2 were less than 0 kcal/mol.In vivo experiment results showed that compared with RSV group,the counts of white blood cells and lymphocytes increased and the lung index decreased in high-dose Yunshi Ganmao Heji group,with statistically significant difference(P<0.05).HE staining showed pulmonary hyperplasia,thickened alveolar wall and in

关 键 词：云实感冒合剂 网络药理学 呼吸道合胞病毒 体内试验 分子对接 

分 类 号：R285[医药卫生—中药学]