药理剂量维生素C杀灭流感病毒的体外研究

An in vitro study on the pharmacological ascorbate treatment of influenza virus

目的探讨药理剂量维生素C体外杀灭流感病毒A／CA／7／09（H1N12009）的作用及其机制。方法将流感病毒A／CA／7／09感染人类正常呼吸道上皮细胞（NHBE）后，立即或8h后分别加入2．5～20mmol／L维生素C、维生素C＋过氧化氢酶和不含维生素C的NHBE培养基，或直接在流感病毒A／CA／7／09中分别加入上述培养基。培养4—12h后，分别收集培养基上清液，通过半数组织培养感染剂量（TCID。）法对病毒进行定量检测。利用高压液相层析法（HPLC）测量样品中维生素C含量和Clark型氧电极法测量样品中过氧化氢浓度。结果药理剂量的维生素C可杀灭体外游离的流感病毒，也能杀灭体外培养细胞内的流感病毒；维生素C杀灭病毒的效应与剂量相关：2．5mmol／L的维生素C可杀灭约90％的流感病毒，20mmol／L的维生素C可杀灭所有病毒；维生素C在病毒感染不同时间窗的杀灭病毒效应不同，在病毒感染8～12h内，维生素C杀灭病毒的效果最好；4h内可清除感染NHBE细胞的所有流感病毒；感染后期维生素C杀灭病毒的效果最差。维生素C杀灭病毒的效应可被过氧化氢酶完全抑制。结论药理剂量的维生素C作为一种强氧化剂，可能通过生成过氧化氢发挥杀灭细胞内外流感病毒的作用。

Objective To investigate the effects and mechanism of pharmacological ascorbate against Influenza A/CA/7/09 （H1N12009）. Methods NHBE cells （ ≈95% confluent monolayer） in 12- well plates （Coming） were kept at 37 ℃ at all times. NHBE cells were exposed to A/CA/7/09 （H1N12009） influenza virus at MOI of 0.01 for 1 h, rinsed with NHBE medium, and incubated with NHBE medium containing 20 mmol/L ascorbate or 20 mmol/L ascorbate ＋ 600 IU/ml Catalase. The ceils were then incubated for an additional 4 - 12 h and the culture medium was harvested for titration. Viral titers were determined as loglo 50% tissue culture infective doses （TCIDs0） assay in MDCK cells. Ascorbate in NHBE medium was determined using HPLC separation coupled with coulometrie electrochemical detection. Hydrogen peroxide was detected indirectly by Clark-type oxygen electrode. Results In vitro experiments showed that pharmacological ascorbate killed not only isolated viruses, but also viruses from normal human bronchial epithelial cells. The antiviral effect of ascorbic acid appeared to be dose-dependent. 2. 5 mmol/L ascorbic acid was able to eliminate 90% of the viruses and 20 mmol/L ascorbic acid totally blocked viral replication in vitro. The antiviral effect of pharmacological aseorbate varied at different phases of infection. Pharmacological aseorbate eliminated viral infectivity with treatment times as short as 4 hours at early stage of infection. But the effect was reversed by eatalase. Conclusion Pharmacological aseorbate （ vitamin C） as a pro-drug eliminates or kills influenza virus, probable by producing steady-state concentrations of hydrogen peroxide （ H2 02 ） in extracellular fluid.