摘要
目的研究槲皮素体外抗呼吸道合胞病毒(respiratory syncytial virus,RSV)的作用,为新型抗呼吸道合胞病毒中药开发提供基础研究数据。方法根据细胞毒性实验确定槲皮素的最大工作浓度,采用病毒滴度TCID50检测法、细胞毒性检测法、Western blot和实时荧光定量PCR(qPCR)方法检测槲皮素体外对RSV的增殖、蛋白表达和基因转录抑制效果。采用RNA干扰敲低HSP70表达水平,检测病毒的生长曲线。结果槲皮素对Vero细胞的毒性呈现明显的梯度依赖效应,最大无毒浓度为25μM,且12μM的槲皮素浓度下,细胞上清中病毒滴度抑制率达60%;同时Western blot结果显示槲皮素处理的细胞中病毒F蛋白受到显著抑制;qPCR对细胞中RSV G蛋白的转录本的检测结果表明槲皮素使病毒G蛋白的转录效率下降了85%以上。敲低HSP70的表达水平后,病毒的滴度下降了两个数量级。结论槲皮素体外对RSV-A有较好的抗病毒作用,且可能是通过抑制其特异性底物HSP70实现的。
Objective To study the effect of quercetin on respiratory syncytial virus(RSV)in vitro,so as to provide basic data for the development of new traditional Chinese medicine against respiratory syncytial virus.Methods According to the maximum working concentration of quercetin determined by cytotoxcity assay,the in vitro effects of quercetin on RSV proliferation,protein expression and gene transcriptional inhibition were analyzed by TCID50 assay,cytotoxicity assay,Western blot and real-time fluorescent quantitative PCR(qPCR).The growth curve of the virus was tested using RNAi to knock-down the expression of Hsp70.Results Toxicity of quercetin on Vero cells showed an obvious gradient dependent effect,with the maximum non-toxic concentration of 25μM.The inhibition rate of virus titer in the cells supernatant reached 60%at the quercetin concentration of 12μM.Meanwhile,Western blot results showed that the virus F protein in cells treated with quercetin was significantly inhibited.The results of qPCR on the transcript of RSV G protein in cells showed that quercetin reduced the transcription efficiency of virus G protein by more than 85%.Knocking down the expression level of HSP70 reduced the titer of the virus by two orders of magnitude.Conclusions Quercetin has a good antiviral effect on RSV-A in vitro,and may be achieved by inhibiting its specific substrate HSP70.
作者
邱艳
周晓蕾
桂碧玉
李艳娟
邓翔
马东波
汪洋
Qiu Yan;Zhou Xiaolei;Gui Biyu;Li Yanjuan;Deng Xiang;Ma Dongbo;Wang Yang(Sixth Respiratory Ward,Xinyang Fifth People's Hospital,Xinyang 464000,China;Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450003,China;Third Respiratory Ward,Henan Chest Hospital,Zhengzhou 450003,China)
出处
《国际病毒学杂志》
2020年第5期372-376,共5页
International Journal of Virology
基金
郑州大学第一附属医院青年基金项目(YNQN2017050)。
关键词
槲皮素
呼吸道合胞病毒
抗病毒作用
Quercetin
Respiratory syncytial virus
Antiviral effect
作者简介
通信作者:汪洋,Email:yangwangmd@163.com,电话:0371-66271102。
