银花平感颗粒的体外抗甲型H1N1流感病毒作用

Anti influenza A (H1N1) Virus Effect of Yinhua Pinggan Keli(银花平感颗粒) : in vitro Study

目的探讨银花平感颗粒体外抗甲型A/PR8/34(H1N1)流感病毒的作用机制。方法采用MTT比色法测定药物对巨噬细胞的最大无毒浓度(TC_0),半数细胞毒性浓度(TC_(50))及药物不同给药方式的抗病毒有效率,采用Probit回归分析计算其对病毒感染的半数抑制浓度(IC_(50))及治疗指数(TI);流感病毒H1N1感染培养的RAW264.7细胞,用利巴韦林(62.5μg/ml),银花平感颗粒高(500μg/ml)、中(250μg/ml)、低(125μg/ml)各剂量进行干预,4、16、24 h后收集细胞,实时荧光定量PCR法观察各组细胞中Toll样受体7(TLR7)、髓样分化因子88(My D88)、核因子p65(NF-κB p65)、干扰素调节因子7(IRF7)的变化。结果银花平感颗粒在31.25~500μg/ml范围内可降低流感病毒复制,其中药物治疗组在给药24 h后,抗病毒有效率最高;与正常对照组同时间比较,模型对照组TLR7、My D88、IRF7、NF-κB p65 mRNA表达水平均显著升高,且16 h达最高峰,较本组4 h、24 h差异均有统计学意义(P<0.05或P<0.01);经药物干预后,银花平感颗粒高、中剂量组及利巴韦林组均显著下调流感病毒诱导4、16、24 h后的TLR7、My D88、IRF7和NF-κB p65 mRNA表达水平(P<0.05或P<0.01),尤以银花平感颗粒高剂量组对TLR7、My D88和NF-κB p65 mRNA表达水平的下调作用较利巴韦林组更为显著(P<0.05)。结论银花平感颗粒体外可明显抑制流感病毒复制,主要是通过治疗给药而发挥抗病毒作用,其抗病毒效果可能与其抑制TLR7/My D88/IRF7信号通路及NF-κB p65的激活有关,且高剂量对流感的治疗效果优于利巴韦林。

Objective To explore the mechanism of Yinhua Pinggan Keli（ 银花平感颗粒） in anti influenza A/PR8/34（ H1N1） virus in vitro. Methods MTT method was used to detecte maximum non-toxic concentration of drugs against macrophages（ TC_0）,median toxic concentration against cells（ TC_（50）） and antiviral efficiency of different drug administration methods. Half inhibited concentration（ IC_（50）） and treatment index（ TI） in virus infection were calculated by probit regression analysis. RAW264. 7 cells infected by influenza H1 N1 virus were intervened by ribavirin（ 62. 5 μg/ml）,Yinhua Pinggan Keli of high（ 500 μg/ml）,medium（ 250 μg/ml） and low（ 125 μg/ml）dose. After 4,16 and 24 hours,the cells were collected. Changes of Toll-like receptor 7（ TLR7）,myeloid differentiation factor 88（ My D88）,nuclear factor p65（ NF-κB p65）,interferon regulatory factor 7（ IRF7） of cells in all groups were examined by real time fluorescent quantitative polymerase chain reaction（ PCR） method. Results Yinhua Pinggan Keli of 31. 25 to 500 μg/ml might reduce influenza virus replication. The antiviral effective rate in the medication group was the highest among all groups after administration for 24 h. Compared with the normal control group at the same time point,the expressions of TLR7,My D88,IRF7 and NF-κB p65 mRNA in the model control group increased,with peak at 16 h. The difference was significant compared with 4 h and 24 h in the same group（ P〈0. 05 or P〈0. 01）. After medicine intervention,the expression levels of TLR7,My D88,IRF7 and NF-κB p65 mRNA induced by influenza virus for 4,16 and 24 h were down-regulated in the Yinhua Pinggan Keli high,medium dose groups and the ribavirin group（ P〈0. 05 or P〈0. 01）. Especially,the down-regulation effect on the expressions of TLR7,My D88 and NF-κB p65 mRNA in the Yinhua Pinggan Keli high dose group was superior to that in the ribavirin group. Conclusion Yinhua Pinggan Keli in vitro might inhibit influenza virus replication. The anti-virus effect was mainly by therapeutic medication,which might relate to inhibiting TLR7/My D88/IRF7 signaling pathway and activation of NF-κB p65. The treatment effect on influenza of high dose was superior to ribavirin.