摘要
【目的】研究异绿原酸A(isochlorogenic acid A,IAA)体外抗小反刍兽疫病毒(peste des petits ruminant virus,PPRV)的效果及其作用机制。【方法】采用MTT法测定IAA对非洲绿猴肾细胞(verda reno,Vero)的最大无毒质量浓度、半数细胞毒性质量浓度(50%cytotoxic mass concentration,CC_(50));空斑形成试验测定IAA的半数有效质量浓度(median effect mass concentration,EC_(50))并计算治疗指数(therapeutic index,TI);MTT法观察IAA对PPRV感染的Vero细胞活性的影响;采用空斑形成试验、实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)、免疫印迹试验和间接免疫荧光试验观察IAA对PPRV增殖和分布的影响;采用活性氧(reactive oxygen species,ROS)荧光探针、硫代巴比妥酸(thiobarbituric acid,TBA)比色法和羟胺比色法分别检测IAA对PPRV感染细胞内ROS、丙二醛(malondialdehyde,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)活性的影响;采用免疫印迹试验检测IAA对PPRV感染细胞中磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、磷酸化AKT(p-AKT)、B细胞淋巴瘤-2蛋白(B cell lymphoma-2 protein,Bcl-2)和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)表达水平的影响。【结果】IAA对Vero细胞的最大无毒质量浓度、CC_(50)、EC_(50)分别为7.81、125.60、21.02 mg·L^(-1),TI值为5.98;IAA处理能显著抑制PPRV诱导的细胞病变及病毒在Vero细胞中的增殖;细胞活力显著高于病毒感染细胞;病毒噬斑形成数量、病毒F和N基因表达水平显著降低;宿主细胞胞浆内病毒分布显著减少。IAA能显著提高PPRV感染细胞SOD活性,清除病毒感染细胞内的ROS和MDA。经IAA处理的PPRV感染细胞中PI3K、AKT表达水平极显著提高,p-AKT表达水平显著降低,Bax/Bcl-2比值显著降低。【结论】IAA通过调节宿主细胞PI3K/AKT通路及其下游细胞凋亡相关蛋白Bax和Bcl-2的表达水平来缓解PPRV感染引起的氧化应激反应,从而�
【Objective】The present work was conducted to investigate the antiviral activity of isochlorogenic acid A(IAA)against peste des petits ruminant virus(PPRV)in vitro and its mechanism.【Methods】The maximum non-toxic mass concentration and the 50%cytotoxic mass concentration(CC_(50))of IAA on African green monkey kidney cells(verda reno,Vero)were determined by MTT method.The median effective mass concentration(EC_(50))of IAA was determined by plaque formation test and the therapeutic index(TI)was calculated.The effect of IAA on the cell viability of the PPRV-infected Vero cells was determined by MTT assay,and the effects of IAA on PPRV proliferation and distribution in the host cells were observed by plaque formation experiment,quantitative real-time PCR(qRT-PCR),western-blot(WB)and indirect immunofluorescen assay respectively.The effects of IAA on the intracellular reactive oxygen species(ROS)content,malondialdehyde content and activity of superoxide dismutase(SOD)of the virus-infected cells were respectively evaluated by fluorescent probe,thiobarbituric acid and hydroxylamine colorimetry method,and the effects of IAA on the expression levels of PI3K,AKT,p-AKT,Bcl-2 and Bax in the PPRV-infected cells were detected by Western-blot.【Result】The results showed that the maximum non-toxic mass concentration,the CC_(50)on Vero cells,the EC_(50)on PPRV and the TI were 7.81,125.60,21.02 mg·L^(-1)and 5.98,respectively.The cytopathic effect induced by PPRV infection was significantly inhibited by IAA treatment and the viability of the virus infected cells was significantly higher than that of PPRV-infected cells.The number of plaque formation units and the expression levels of viral structural protein F and N were significantly decreased.The virions in cytoplasm of vero cells were also decreased significantly.Moreover,the activity of SOD of the PPRV-infected cells was significantly increased by IAA,and the content of ROS and MDA was accordingly decreased.The expression levels of PI3K and AKT were significantly up-r
作者
焦豪杰
陈绍红
刘铀
JIAO Haojie;CHEN Shaohong;LIU You(College of Coastal Agricultural Science,Guangdong Ocean University,Zhanjiang 524088,China;College of Food Science and Technology,Guangdong Ocean University,Zhanjiang 524088,China)
出处
《河南农业大学学报》
CAS
CSCD
2023年第1期125-135,共11页
Journal of Henan Agricultural University
基金
广东省现代农业产业技术体系南方现代草牧业产业项目(2019K127)。
关键词
异绿原酸A
小反刍兽疫病毒
抗病毒活性
作用机制
信号通路
isochlorogenic acid A
peste des petis ruminants virus
antiviral activity
action mechanism
signal pathway
