目的 通過構(gòu)建分子網(wǎng)絡，結(jié)合分子對接與實驗驗證探究清咽滴丸抗流感的活性物質(zhì)。方法 用噻唑藍（MTT）法檢測不同濃度清咽滴丸對非洲綠猴腎細胞Vero E6細胞活力的影響，確定安全劑量。以甲型H1N1流感病毒（H1N1）毒株P(guān)R8構(gòu)建病毒感染Vero E6細胞模型，評價清咽滴丸的體外抗病毒活性。通過中藥系統(tǒng)藥理學數(shù)據(jù)庫與分析平臺（TCMSP）、PubChem數(shù)據(jù)庫獲取清咽滴丸活性成分及其作用靶點，以GeneCards、OMIM、DrugBank和Swiss Target Prediction數(shù)據(jù)庫獲取流感靶點，經(jīng)Uniprot數(shù)據(jù)庫進行蛋白-基因的標準化轉(zhuǎn)換，以活性成分靶點與疾病靶點取交集繪制Venn圖，基于Cytoscape軟件構(gòu)建“中藥-活性成分-關(guān)鍵靶點-疾病”分子網(wǎng)絡，通過STRING數(shù)據(jù)庫搭建蛋白質(zhì)-蛋白質(zhì)相互作用（PPI）網(wǎng)絡，采用DAVID數(shù)據(jù)庫進行基因本體（GO）和京都基因與基因組百科全書（KEGG）通路富集分析。通過BIOVIA Discovery Studio2020軟件計算系統(tǒng)進行潛在活性成分和關(guān)鍵靶點的分子對接，基于表面等離子共振技術(shù)（SPR）對虛擬篩選結(jié)果進行驗證，闡釋活性分子與靶點的親和活性，并通過ELISA方法檢測了白細胞介素-6（IL-6）、腫瘤壞死因子-α（TNF-α）的表達水平，評價活性分子的抗炎活性。結(jié)果體外實驗結(jié)果表明清咽滴丸對H1N1甲型流感病毒株P(guān)R8在質(zhì)量濃度為500 μg·mL^-1時，對流感病毒抑制率為49.70%。清咽滴丸抗流感的潛在活性成分118個，共有靶點94個，度值排名前10位的核心靶點為IL-6、TNF、腫瘤蛋白p53（TP53）、前列腺素內(nèi)過氧化物合酶2（PTGS2）、IL-1β、胱天蛋白酶3（CASP3）、原癌基因（JUN）、骨髓細胞瘤癌基因（MYC）、IL-10、白細胞介素-8（CXCL8），其主要通過調(diào)節(jié)IL-17信號通路、TNF信號通路等發(fā)揮抗流感作用。組方中的異鼠李素、山柰酚、訶子次酸和鞣花酸與關(guān)鍵靶點IL-6與TNF-α具有良好的親和活力，并具有顯著的抗炎作用，提示其可能是組方發(fā)揮抗流感的潛在活性成分。結(jié)論 清咽滴丸通過異鼠李素、山柰酚、訶子次酸和鞣花酸等活性物質(zhì)調(diào)節(jié)機體免疫功能、作用于細胞因子以及調(diào)控炎癥相關(guān)信號通路，協(xié)同發(fā)揮抗流感的作用。

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

天津市教委科研計劃項目（ 2019KJ178）