升降散体外抗流感病毒A/PR8/34/H1N1株的作用研究

Antiviral Effect of Shengjiang Powder on Influenza Virus A/PR8/34/HIN1 in Vitro

目的研究升降散体外抗流感病毒A/PR8/34/H1N1株的作用。方法以磷酸奥司他韦作为阳性对照药物,在狗肾细胞株水平上,测定流感病毒的病毒滴度,计算流感病毒的半数细胞培养感染剂量(TCID_(50));测定阳性对照药和升降散的细胞毒性,得到药物的最大无毒浓度(TC_(0))和半数中毒浓度(TC_(50));通过3种不同的给药方式体外观察升降散对甲型流感病毒PR8感染的细胞病变效应(CPE)的作用影响,采用CCK8法计算升降散对甲型流感病毒PR8感染的细胞存活率的影响,计算药物的抗病毒有效率(ER);同时采用实时荧光定量PCR法在给药后12、24、48h动态检测细胞内Caspase-3、Caspase-8、Caspase-9凋亡相关因子的表达水平,探讨升降散抗病毒作用机制。结果病毒滴度测定流感病毒PR8的TCID_(50)为10^(-5.20);药物毒性测定得到磷酸奥司他韦和升降散的TC_(0)分别为0.25、6.25mg/mL,TC_(50)分别为0.76、9.69mg/mL;3种给药方式下的升降散组和磷酸奥司他韦组的细胞存活率高于病毒对照组(P<0.05),在最大无毒浓度下升降散直接灭活抗流感病毒的ER为52.37%,通过预防病毒侵入细胞来抗病毒的ER和治疗病毒穿入细胞来抗病毒的ER低于50%;升降散组细胞在12、24、48 h的Caspase-3、Caspase-8、Caspase-9基因mRNA表达均被显著下调(P<0.05)。结论升降散具有一定的体外抗流感病毒作用,且能通过抑制流感病毒感染诱导的细胞凋亡起作用。

Objective:To investigate the antiviral effect of Shengjiang Powder on influenza virus A/PR8/34/HIN1 in vitro.Methods:MDCK cells were used as host cells and Oseltamivir phosphate as positive drug,the virus titer was determined and the half tissue culture infection dose(TCID_(50))of influenza virus was calculated.The cytotoxicity of positive control drug and Shengjiang Powder was determined,and the maximum non-toxic concentration(TC_(0))and half toxic concentration(TC_(50))were obtained.Under three different administration methods,the effect of Shengjiang Powder on the cytopathic effect(CPE)of influenza A virus PR8 infection was observed in vitro.The effect of Shengjiang Powder on the cell survival rate of influenza A virus PR8 infection was calculated by CCK8 method,and the antiviral efficacy(ER)of the drug was calculated.At the same time,the expression levels of apoptosis-related factors Caspase-3,8 and 9 were dynamically detected by real-time fluorescence quantitative PCR at 12 h,24 h and 48 h after administration to explore the antiviral mechanism of Shengjiang Powder.Results:The TCID_(50) of influenza virus PR8 was 10^(-5.20).The TC_(0) of oseltamivir phosphate and Shengjiang Powder were 0.25 mg/mL and 6.25 mg/mL,and the TC_(50)of oseltamivir phosphate and Shengjiang Powder were 0.76 mg/mL and 19.69 mg/mL.The cell survival rate of Shengjiang Powder group and oseltamivir phosphate group were higher than that of virus control group,and the difference was statistically significant(P<0.05).Under the maximum non-toxic concentration,the ER of Shengjiang Powder directly inactivated anti-influenza virus was 52.37%,which of anti-virus by preventing virus invasion and treating virus penetration into cells was less than 50%.The mRNA expressions of Caspase-3,Caspase-8 and Caspase-9 genes in Shengjiang Powder group were significantly down-regulated at 12 h,24 h and 48 h,and the difference was statistically significant(P<0.05).Conclusion:Shengjiang Powder has a certain anti-influenza virus effect in vitro,and has inhibit effect the apoptosis induced by influenza virus infection.