银花青蒿栀子方抗A6型柯萨奇病毒的作用

Antiviral Effect of YQZ Against Coxsackievirus A6

A6型柯萨奇病毒(Coxsackievirus A6,CV⁃A6)感染致手足口病发病率呈上升趋势,逐渐成为部分地区的主要流行株。但是,CV⁃A6的相关研究目前较少,亟需抗CV⁃A6药物的研究。中药在抗病毒方面具有独特优势,银花青蒿栀子方(Yinhua⁃Qinghao⁃Zhizi,YQZ)对多种病毒有效,但对CV⁃A6的作用尚未见研究报道,本研究应用细胞和动物感染模型评价了YQZ体内外抗CV⁃A6的作用。体外实验中,利用CV⁃A6感染RD细胞模型,以病毒抑制率和细胞上清病毒载量为指标评价YQZ的抗病毒作用,并采用Box–Behnken设计⁃响应面法优化YQZ的配伍比例。体内实验中,10日龄ICR乳鼠按窝随机分为正常对照组、模型组、阳性对照组(利巴韦林10 mg/kg)、低剂量YQZ组(生药1.3g/kg)和高剂量YQZ组(生药2.6 g/kg)。采用滴度为107.4 TCID50/mL的CV⁃A6 ip感染制备模型,统计生存率和临床评分;取骨骼肌、心脏、肺脏、脊髓、延髓等组织,HE染色观察病理变化;一步法RT⁃qPCR检测上述各组织的病毒载量。结果显示,YQZ抗CV⁃A6的最佳配伍为:金银花3 g、青蒿15 g、栀子2 g,其病毒抑制率和病毒载量拷贝数的对数均与模型预测值接近(P>0.05),且活性显著优于对照利巴韦林(P<0.01)。与模型组相比,低、高剂量YQZ组乳鼠生存率提高,急性弛缓性麻痹等症状明显缓解,骨骼肌、心脏、肺脏等脏器的病理损伤明显减轻,上述组织病毒载量均下降,其中高剂量YQZ组乳鼠骨骼肌、心脏和肺组织中的病毒载量显著降低(P<0.05)。以上结果表明,Box–Behnken设计⁃响应面法可促进YQZ的配伍优化筛选、提高药效,优化配伍比例的YQZ具有良好的体内外抗CV⁃A6作用。

The incidence of hand,foot and mouth disease(HFMD)caused by Coxsackievirus A6(CV⁃A6)infection has been increasing,and HFMD has became an epidemic in parts of China.Few relevant studies on CV⁃A6 have been completed.Traditional Chinese medicine has unique advantages for the prevention and treatment of viral diseases.It has been reported that Yinhua⁃Qinghao⁃Zhizi(YQZ)has effect against several types of virus,not including CV⁃A6.We investigated the in vitro and in vivo effects of YQZ against CV⁃A6 with cell models and animal–infection models,respectively.In in vitro experiments,the compatibility of YQZ against CV⁃A6 was optimized by Box⁃Behnken design–response surface methodology(BBD–RSM)using virus⁃infected RD cells with percent inhibition of virus and viral load in cell supernatants as dependent variables.In in vivo experiments,10⁃day⁃old ICR mice were divided into five groups randomly:normal control group,model group,positive control group(ribavirin 10 mg/kg),low–dose YQZ group(1.3 g/kg of crude material),and high–dose YQZ group(2.6 g/kg of crude material).Except normal control group,mice in other groups were injected intraperitoneally(i.p.)with 107.4 TCID50/mL of CV⁃A6.Percent survival and clinical score were recorded,tissues(skeletal muscle,heart,lung,spinal cord,and medulla)were collected and pathology conducted using H&E staining.The viral load in tissues was detected by one step real time reverse transcription⁃quantitative polymerase chain reaction.The optimal compatibility of YQZ against CV⁃A6 was:Lonicerae japonicae 3 g,Artemisia annua 15 g and Gardeniae Fructus 2 g.Percent inhibition of virus and copy logarithm of virus load were close to the predicted values(P>0.05),respectively,and the antiviral activity of YQZ was significantly superior compared with that of ribavirin(P<0.01).Compared with the model group,the percentage of mice that survived in the low–dose YQZ group and high–dose YQZ group was higher and symptoms e.g.acute flaccid paralysis were relieved.The tissue injuries were relieved markedly,and the viral load in various tissues was decreased.The decrease in injury to skeletal muscle,heart and lungs in the high–dose YQZ group was significant(P<0.05).These results indicated that application of BBD–RSM could facilitate optimization of compatibility and potency enhancement of YQZ,and that optimized YQZ exhibited potent activity against CV⁃A6 in vitro and in vivo.