氧化苦参碱抗流感病毒药效及机制研究

The effects and mechanisms of oxymatrine on anti-influenza A virus activity

目的：研究氧化苦参碱的抗流感病毒活性及其潜在机制。方法：培养MDCK和A549细胞，并设空白对照组（DMSO，0.5%）、病毒处理组、阳性对照组（利巴韦林，200μmol/L）和氧化苦参碱处理组（12.5、25、50、100 mol/L），每组重复6孔。空斑抑制法测定氧化苦参碱抗病毒药效；构建荧光素酶报告质粒，并用Western blot和ELISA检测氧化苦参碱对流感病毒诱导的细胞Toll样受体4（TLR4）、髓样分化因子88（MyD88）和肿瘤坏死因子受体相关因子6（TRAF6）启动子转录、TLR4-Myd88-TRAF6-NF-κB信号通路活化及炎性因子释放的影响。结果：空斑抑制实验显示氧化苦参碱在12.5～100μmol/L范围内可显著降低流感病毒复制，其半数有效浓度（EC ）为19.95μmol/L。启动子荧光素酶报告质粒检测显示12.5μmol/L氧化苦参碱可显著降低流感病毒诱50导的TLR4、MyD88和TRAF6转录（P＜0.05）；Western blot检测显示12.5μmol/L氧化苦参碱可显著降低流感病毒诱导的TLR4-Myd88-TRAF6-NF-κB通路的活化（P＜0.05）；ELISA检测显示12.5μmol/L氧化苦参碱可显著降低流感病毒诱导的IL-1β、IL-6、TNF-α、IL-8、IFN-β和IFN-γ释放（P＜0.05）。结论：氧化苦参碱具抗流感病毒活性，其潜在机制可能与其抑制流感病毒诱导的TLR4-Myd88-TRAF6-NF-κB信号通路活化有关。μ与病毒处理组比较，

OBJECTIVE： To explore the effects and mechanisms of oxymatrine on anti-influenza A virus （IAV） activity. METHODS： MDCK and A549 cells were cultured and divided into 4 groups： blank control （DMSO, 0.5%）, virus-treated group,positive group （ribavirin,200 μmol/L） and oxymatrine-treated group （12.5,25,50 and 100 μmol/L） ,with 6 repeats in each group. The anti-IAV activity of oxymatrine was determined by plaque inhibition assay. The influence of oxymatrine on the IAV-induced transcription of TLR4,MyD88 and TRAF6,the activation of TLR4-Myd88-TRAF6-NF-κB signal pathway,and the release of inflammatory cytokins were determined by luciferase reporter plasmid,Western blot and ELISA assays,respectively. RESULTS： Plaque inhibition assay showed that oxymatrine,at a range of 12.5-100μmol/L,could significantly inhibit the replication of IAV in vitro,and its EC50 was 19.95μmol/L. Luciferase reporter assay showed that oxymatrine,at the concentration of 12.5μmol/L,could significantly inhibit the transcription of TLR4,MyD88 and TRAF6 induced by IAV infection （compared with virus-treated group,P〈0.05）. Western blot assay showed that oxymatrine （12.5μmol/L） could significantly inhibit the IAV-induced activation of TLR4-Myd88-TRAF6-NF-κB pathway （compared with virus-treated group,P〈0.05）. ELISA assay showed that oxymatrine （12.5 μmol/L） could significantly inhibit the IAV-induced release of inflammatory cytokins （compared with virus-treated group,P〈0.05）. CONCLUSION： Oxymatrine possessed anti-IAV activity,and its mechanism may be related to its ability to inhibit the IAV-induced activation of TLR4-MyD88-TRAF6-NF-κB pathway.