金莲清热泡腾片抗流感病毒体内外药效实验及作用机制研究

Study on in vitro and in vivo pharmacodynamic tests of Jinlian Qingre Effervescent Tablets against influenza virus and its mechanism

目的研究金莲清热泡腾片体内外抗流感病毒的药效学及作用机制。方法进行体外实验考察金莲清热泡腾片对甲1型流感病毒株(NYMCX-179A)、甲3型流感病毒株(NYMCX-327)和乙型流感病毒株(B/Phuket/3073/2013)的作用,采用细胞病变效应(CPE)法检测高、中、低3个浓度下药物有效浓度(EC_(50)),以及在细胞病毒感染后的不同时间点药物的作用情况及病毒滴度(TCID_(50))。进行体内实验考察金莲清热泡腾片对甲1型流感病毒(鸡胚扩增)的作用,计算小鼠存活率、生存时间;观察肺组织切片;CPE法、四甲基偶氮唑盐微量酶反应比色(MTT)法、血球凝集(HA)法测定其TCID_(50)。蛋白免疫印迹技术(WB)检测肺组织中H1N1病毒血凝素(HA)蛋白,探讨抗病毒机制。结果在体外实验中,预防模式用药和治疗模式用药的金莲清热泡腾片高、中、低3个浓度均能明显抑制甲1型流感病毒10 TCID_(50)、甲3型流感病毒10 TCID_(50)、乙型流感病毒株100 TCID_(50)引起的细胞病变,其中金莲清热泡腾片高浓度组效果优于中、低浓度;细胞感染甲1型流感病毒后,0、2、4、6、8、10、12 h给药(最大有效浓度为5 mg/ml),显示金莲清热泡腾片均可在流感病毒复制的1个周期内有效抑制病毒的复制,子代病毒产量显著降低;体内抗甲1型流感病毒实验结果显示,金莲清热泡腾片高、中、低浓度组在治疗初期就有抗H1N1病毒的作用,在第4天后作用更明显,金莲清热泡腾片高浓度组第6天肺内病毒活性基本抑制,肺组织病理损伤基本恢复。WB实验表明该药可抑制H1N1病毒在肺组织中的HA蛋白表达。结论金莲清热泡腾片具有显著的抗流感病毒作用,可用于治疗因病毒引起的流感或上呼吸道疾病。

Objective To study the pharmacodynamics and mechanism of Jinlian Qingre Effervescent Tablets against influenza virus in vitro and in vivo.Methods The effects of Jinlian Qingre Effervescent Tablets on influenza A1 virus(NYMCX-179A),influenza A3 virus(NYMCX-327)and influenza B virus(B/Phuket/3073/2013)were investigated in vitro.The cytopathic effect(CPE)was adopted to detect the effective concentration(EC_(50))of the drug at high,medium and low concentrations,as well as the effect of the drug and viral titer(TCID_(50))at different time points after cell virus infection.The effect of Jinlian Qingre Effervescent Tablets on influenza A1 virus(chick embryo amplification)was investigated in vivo,and the survival rate and survival time of mice were calculated;the lung tissue slices were observed;the TCID_(50)was determined by CPE method,microenzyme reaction colorimetry(methyl thiazolyl tetrazolium[MTT]method),and hemagglutination(HA)method.The hemagglutinin(HA)protein of H1N1 virus in the lung tissue was detected by western blotting(WB)to explore the antiviral mechanism.Results In the in vitro experiments,the high,medium and low concentrations groups of Jinlian Qingre Effervescent Tablets in the preventive mode and therapeutic mode could significantly inhibit the cytopathic effects caused by 10 TCID_(50)of influenza A1 and A3 virus strain,and 100 TCID_(50)of influenza B virus strains,and the effect in the high concentration group of Jinlian Qingre Effervescent Tablets was better than that in the medium concentration group and the low concentration group of Jinlian Qingre Effervescent Tablets.After the cell infection by influenza A1 virus,the drug was administered at 0,2,4,6,8,10 and 12 h(the maximum effective concentration was 5 mg/ml).The results showed that all the effervescent tablets could effectively inhibit virus replication within one cycle of influenza virus replication,and the production of offspring virus was significantly reduced.The experimental results of anti-influenza A1 virus in vivo showed that the high,medium and low concentrations groups of Jinlian Qingre Effervescent Tablets had anti-H1N1 virus effect at the early stage of treatment,which became more significant after the fourth day.The virus activity in the lung of the high concentrations group of Jinlian Qingre Effervescent Tablets was basically inhibited on the sixth day,and the pathological damage in the lung tissue was basically recovered.WB experiments showed that the drug could inhibit the expression of HA protein of H1N1 virus in the lung tissue.Conclusion Jinlian Qingre Effervescent Tablets have significant anti-influenza virus effect,and can be used to treat influenza or upper respiratory tract diseases caused by virus.