乙酰紫草素体外抗2型单纯疱疹病毒机理的研究

Study on antiviral mechanism of acetylshikonin against herpes simplex virus type 2 in vitro

目的研究乙酰紫草素(acetylshikonin,AS)体外抗2型单纯疱疹病毒(herpes simplex virus type 2,HSV-2)的活性及作用机理。方法以阿昔洛韦(acyclovir,ACV)为阳性对照药物,采用CCK-8法检测细胞活力,确定AS对成年非洲绿猴肾细胞(Vero)的最大无毒浓度;致细胞病变法和噬斑形成抑制实验测定AS对HSV-2的抗病毒活性;实时荧光定量PCR及半数组织培养感染剂量(50%tissue culture infective dose,TCID50)法测定病毒载量以评估AS对HSV-2复制周期不同阶段的抑制作用;透射电镜观察AS对HSV-2病毒粒子结构的影响。结果AS对Vero细胞的最大无毒浓度(maximal atoxic concentration,TC0)为3.785μmol/L,浓度为1.678~3.785μmol/L时能有效抑制HSV-2复制,减少噬斑形成,半数有效浓度(50%effective concentration,EC50)为0.334μmol/L。在12和36 h,病毒释放期加入AS的实验组TCID50低于病毒感染对照组(13.43±10.04 vs.127.00±0.32;3.47±0.55 vs.5.80±0.12),差异均有统计学意义(t=8.359、4.161,P均<0.05);在12、24、36 h,AS直接灭活病毒组病毒载量低于病毒感染对照组(0.24±0.09 vs.1.35±0.07;4.46±0.06 vs.6.75±0.04;2.70±0.04 vs.5.27±0.10),差异均有统计学意义(t=9.920、33.360、24.020,P均<0.05)。此外,AS处理可导致HSV-2病毒粒子形态异常,随着AS浓度升高,作用后的HSV-2超微结构发生渐进改变。结论AS对HSV-2具有一定抗病毒效应,其机理可能为直接破坏病毒颗粒完整结构发挥抗病毒作用。

Objective To investigate the activity and mechanism of acetylshikonin(AS)against herpes simplex virus type 2(HSV-2).Methods By acyclovir(ACV)being used as positive control agent,we examined the cell viability with CCK-8 in order to determine the maximum safe concentration of AS to Vero cell.Cytopathogenic effect method and plaque-reduction neutralization assay were used to detect the antiviral activity of AS against HSV-2.RT-qPCR incubated with TCID50 was used to determine viral load to evaluate the inhibitory effect of AS on different stages of HSV-2 replication cycle.The effect of AS on virion morphology was visualized under the transmission electron microscope.Results The maximal atoxic concentration of AS to Vero cell was 3.785μmol/L,and AS significantly inhibited the replication of virus and reduced plaque formation at the safe concentration of 1.678-3.785μmol/L.Meanwhile,the replication of HSV-2 virus was inhibited by AS with an 50%effective concentration(EC50)value at 0.334μmol/L.The virus titer of the treatment group was lower than that of the corresponding virus infection control group at 12 h and 36 h of release stage(13.43±10.04 vs.127.00±0.32,3.47±0.55 vs.5.80±0.12,respectively),the difference was statistically significant(t=8.359,4.161;both P<0.05),and the virus replication load of the treatment group was lower than that of the corresponding virus infection control group at 12 h,24 h and 36 h of co-incubation with HSV-2(0.24±0.09 vs.1.35±0.07,4.46±0.06 vs.6.75±0.04,2.70±0.04 vs.5.27±0.10,respectively),the difference was statistically significant(t=9.920,33.360,24.020;all P<0.05).Besides,treatment with AS resulted in abnormal morphology of certain HSV-2 virions,and the ultrastructure of HSV-2 changed gradually with added AS concentration.Conclusion The results reported here indicated that AS possessed a certain antiviral effect on HSV-2.The potential mechanism is to exert antiviral effect by directly destroying the complete structure of the virus particles.