黑色素及谷胱甘肽通过抑制NF-κB途径的激活可有效抑制H 5N1禽流感病毒感染

我国是H5N1高致病性禽流感频发区.为筛选天然药物用于H5N1禽流感的防治并研究其抗病毒感染的作用机制，本研究选取黑色素及谷胱甘肽（GSH）为候选药物，通过Hoechst 33258荧光染色,观察了禽流感病毒(AIV)感染后的细胞形态变化；细胞凋亡流式细胞术定量分析表明，H5N1 AIV感染可诱导宿主狗肾上皮细胞(MDCK)凋亡，而黑色素及GSH对其具有显著抑制作用(P<0.01)；当20 μg/ml黑色素及10 mmol/LGSH共同作用时,抑制率高达89% (P <0.01)；采用MTT法检测,黑色素及GSH可增强AIV感染后的宿主细胞存活率；病毒血凝素(Ｈa)基因的RT-PCR检测结果表明，黑色素及GSH可显著降低AIV在宿主细胞内的增殖(P<0.01)；一氧化氮（NO）毛细管电泳（CE）激光诱导荧光检测显示，黑色素及GSH均可显著降低病毒感染诱导宿主细胞内的NO释放量(P<0.05)；RT-PCR技术结合ELISA方法,分别分析了病毒感染后宿主细胞的诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、环氧化酶(cyclooxygenase-2, COX-2)及核因子κB(nuclear factor-kappa B，NF-κB）在mRNA和蛋白水平表达的差异.结果表明，黑色素及GSH可抑制病毒感染诱导的iNOS与COX-2的基因及蛋白的表达 (P<0.05)，并进而抑制NF-κB途径的激活(P<0.01)，从而可有效抑制H5N1 AIV感染.

Inhibition of H5N1 Avian Influenza Virus Infection by Melanin and Glutathione Through Inhibiting the Activation of NF-κB Pathway

China is among the high-occurrence regions of high-pathogenic H5N1 avian influenza virus (AIV) in the world. This study is to screen for the natural antiviral agents for the prevention of AIV and explore the underlined mechanisms. Melanin and glutathione (GSH) were selected as the anti-viral candidates, and their effects on viral-induced apoptosis were evaluated by cytological observation and quantitative flow cytometry analyses. The results showed that the maximum inhibition of viral proliferation in MDCK cells was 89% (P<0.01) with 20 μg/mlmelanin and 10 mmol/L GSH nalyzed by semi-quantitative RT-PCR. The MTT cell viability assay showed that melanin and GSH protected the host cells against H5N1 virus infection (P< 0.01). The quantitation of viral-induced nitric oxide (NO) release was determined by capillary electrophoresis (CE) with laser-induced fluorescence (LIF), and the data exhibited a significant reduction in melanin and GSH-treated MDCK cells. The expressions of inducible nitric oxide synthase (iNOS),cyclooxygenase II (COX-2) and nuclear factor-kappa B (NF-κB) at mRNA and protein levels were assayed by RT-PCR and enzyme-linked immunosorbent assays (ELISA)The results showed that the iNOS and COX-2 activities were decreased (P< 005) and the activation of NF-κB pathway was significantly inhibited (P<0.01) withmelanin and GSH individually or combined. The study suggested that the inhibition of H5N1 AIV infection with melanin and GSH in MDCK cells might be through the inhibition of the viral-induced activation of NF-κB pathway.