目的 通过构建分子网络，结合分子对接与实验验证探究清咽滴丸抗流感的活性物质。方法 用噻唑蓝（MTT）法检测不同浓度清咽滴丸对非洲绿猴肾细胞Vero E6细胞活力的影响，确定安全剂量。以甲型H1N1流感病毒（H1N1）毒株PR8构建病毒感染Vero E6细胞模型，评价清咽滴丸的体外抗病毒活性。通过中药系统药理学数据库与分析平台（TCMSP）、PubChem数据库获取清咽滴丸活性成分及其作用靶点，以GeneCards、OMIM、DrugBank和Swiss Target Prediction数据库获取流感靶点，经Uniprot数据库进行蛋白-基因的标准化转换，以活性成分靶点与疾病靶点取交集绘制Venn图，基于Cytoscape软件构建“中药-活性成分-关键靶点-疾病”分子网络，通过STRING数据库搭建蛋白质-蛋白质相互作用（PPI）网络，采用DAVID数据库进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。通过BIOVIA Discovery Studio2020软件计算系统进行潜在活性成分和关键靶点的分子对接，基于表面等离子共振技术（SPR）对虚拟筛选结果进行验证，阐释活性分子与靶点的亲和活性，并通过ELISA方法检测了白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）的表达水平，评价活性分子的抗炎活性。结果体外实验结果表明清咽滴丸对H1N1甲型流感病毒株PR8在质量浓度为500 μg·mL^-1时，对流感病毒抑制率为49.70%。清咽滴丸抗流感的潜在活性成分118个，共有靶点94个，度值排名前10位的核心靶点为IL-6、TNF、肿瘤蛋白p53（TP53）、前列腺素内过氧化物合酶2（PTGS2）、IL-1β、胱天蛋白酶3（CASP3）、原癌基因（JUN）、骨髓细胞瘤癌基因（MYC）、IL-10、白细胞介素-8（CXCL8），其主要通过调节IL-17信号通路、TNF信号通路等发挥抗流感作用。组方中的异鼠李素、山柰酚、诃子次酸和鞣花酸与关键靶点IL-6与TNF-α具有良好的亲和活力，并具有显著的抗炎作用，提示其可能是组方发挥抗流感的潜在活性成分。结论 清咽滴丸通过异鼠李素、山柰酚、诃子次酸和鞣花酸等活性物质调节机体免疫功能、作用于细胞因子以及调控炎症相关信号通路，协同发挥抗流感的作用。

Objective To unveil the potential active compounds of Qingyan Dropping Pills (QDP) for the treatment of influenza via an integrated strategy combining molecular networks, molecular docking, and experimental verification. Methods First, cell viability was assessed by MTT assay at various concentrations. The H1N1 influenza A virus PR8 strain was used to infect Vero E6 cells for evaluating the antiviral activity. Then, the potential active compounds and targets of QDP were retrieved from the TCMSP and PubChem databases. Disease-related targets of influenza were obtained from GeneCards, OMIM, DrugBank, and Swiss Target Prediction databases. Protein-gene symbol conversion was conducted using the Uniprot database. A Venn diagram was generated to identify the intersection between active compound targets and disease targets. The “Herb-compound-target-disease” network was constructed by Cytoscape software. A protein-protein interaction (PPI) network was generated via STRING database, and Gene Ontology (GO) function as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on key targets using the DAVID database. Potential active compounds were docked with key targets to verify molecular interaction via the BIOVIA Discovery Studio 2020 computational system. Finally, surface plasmon resonance (SPR) assay were explored to verify the binding affinities between active compounds and targets. Additionally, the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA) in vitro to verify the anti-inflammatory activity of potential active compounds. Results QDP were against the H1N1 influenza A virus PR8 strain with the inhibitory rate of 49.70% in vitro. A total of 118 potential active compounds and 94 targets were screened. The top 10 core targets, including IL-6, TNF, tumor protein 53 (TP53), prostaglandin-endoperoxide synthase 2 (PTGS2), IL-1β, caspase-3 (CASP3), Jun proto-oncogene (JUN), myc proto-oncogene (MYC), IL-10, and interleukin-8 (CXCL8), played an important role against anti-influenza virus by regulating the IL-17 signaling pathway and TNF signaling pathway. Isorhamnetin, kaempferol, chebulic acid, and ellagic acid showed good binding affinities with IL-6 and TNF-α, and significantly reduced the levels of inflammatory cytokines (IL-6 and TNF-α). Conclusion Isorhamnetin, kaempferol, chebulic acid, and ellagic acid were verified as the active compounds in QDP to perform anti-influenza activity mainly via regulating immune function, inhibiting inflammatory responses, and modulating cytokines and pathways.

R285.5

天津市教委科研计划项目（ 2019KJ178）