氯化锂体外抗甲型流感病毒H3N2活性研究

In vitro anti-influenza A virus H3N2 activity of lithium chloride

目标分析氯化锂(Lithium chloride,LiCl)在人非小细胞肺癌细胞(A549)抗甲型H3N2流感病毒(Influenza A virus(H3N2))活性研究。方法不同浓度LiCl作用于A549细胞,24 h后观察细胞病变(cytopathic effect,CPE),CCK-8方法测定其对细胞活性的影响;H3N2(MOI=1)感染A549细胞后,加入不同浓度的LiCl作用24 h,观察CPE、RT-qPCR方法测定病毒载量,测定病毒滴度;不同浓度LiCl提前和A549在37℃,5%CO 2孵育2 h,加入病毒感染24 h,RT-qPCR方法测定病毒载量;LiCl、H3N2和A5494℃孵育1 h,35℃,5%CO 2培养24 h,RT-qPCR方法测定病毒载量;H3N2和A5494℃孵育1 h,然后加入不同浓度LiCl,35℃,5%CO 2培养24 h,RT-qPCR方法测定病毒载量;H3N2感染A549细胞后,加入不同浓度的LiCl作用24 h,分别检测上清和细胞的病毒RNA载量和病毒滴度,然后计算上清和细胞相应的比值H3N2(MOI=10)和BV(MOI=1)感染A549细胞后,加入不同浓度的LiCl作用24 h,RT-qPCR方法测定病毒载量。结果LiCl浓度<50 mmol/L时,A549的细胞活性>90%;不同浓度的LiCl均可显著降低H3N2病毒载量(P<0.0001),LiCl处理组的CPE较病毒对照组有所缓解,并且呈剂量依赖性;LiCl不通过影响细胞本身抑制病毒复制;不同浓度LiCl显著抑制H3N2进入A549(P<0.0001)也对吸附A549细胞有一定程度的抑制作用(P<0.01);LiCl不影响H3N2的组装和释放(P>0.05);最后也发现LiCl对多种流感病毒毒株有广谱的抗病毒效果(P<0.0001)。结论LiCl可能通过抑制H3N2吸附和进入A549细胞以及H3N2在A549细胞中复制来发挥抗病毒作用,为LiCl防治病毒感染提供了数据参考。

ObjectiveTo analyze the activity of lithium chloride(LiCl)against influenza virus A(H3N2)in human non-small cell lung cancer cells(A549).MethodsDifferent concentrations of LiCl were incubated with A549 cells,and the cytopathic effect(CPE)was observed after 24 hours,and the effect of LiCl on cell activity was determined by CCK-8 method.After H3N2(MOI=1)infected A549 cells,different concentrations of LiCl were added and incubated for 24 hours,and the viral load was measured by real time/reverse transcription quantitative polymerase chain reaction(RT-qPCR),and the CPE was observed,and the viral titer was determined.Different concentrations of LiCl were incubated with A549 at 37℃and 5%CO 2 for 2 hours,virus was added and incubated for 24 hours,and the viral load was determined by RT-qPCR.LiCl,H3N2 and A549 were incubated at 4℃for 1 hour,35℃,5%CO 2 for 24 hours,and viral load was determined by RT-qPCR.H3N2 and A549 were incubated at 4℃for 1 hour,then different concentrations of LiCl were added,incubated at 35℃with 5%CO 2 for 24 hours,and the viral load was determined by RT-qPCR.After H3N2 infected A549 cells,different concentrations of LiCl were added and incubated for 24 hours,and the viral RNA load and viral titer of the supernatant and cells were measured,respectively,and then the corresponding ratios of the supernatant and the cells were calculated.After H3N2(MOI=10)and BV(MOI=1)infected A549 cells,different concentrations of LiCl were added for 24 h,and the viral load was determined by RT-qPCR.ResultsWhen the concentration of LiCl was<50 mmol/L,the cell viability of A549>90%.Different concentrations of LiCl could significantly reduce the viral load of H3N2(P<0.0001),and the CPE of the LiCl treatment group was more dose-dependent than that of the control group.LiCl did not inhibit viral replication by affecting the cell itself;Different concentrations of LiCl significantly inhibited the entry of H3N2 into A549(P<0.0001),and also had a certain inhibitory effect on the adsorption of A549 cells(P<0.1).LiCl did not affect the assembly and release of H3N2(P>0.05),and it was also found that LiCl had a broad spectrum of antiviral effects against multiple influenza virus strains(P<0.0001).ConclusionsLiCl may exert antiviral effect by inhibiting the adsorption and entry of H3N2 into A549 cells and the replication of H3N2 in A549 cells,which provides a data reference for the prevention and treatment of viral infection by LiCl.