伪狂犬病毒对小鼠肝脏线粒体氧化应激和细胞凋亡影响的研究

Effects of pseudorabies virus infection on mitochondrial oxidative stress and apoptosis in mice liver

为了研究伪狂犬病毒(PRV)感染对小鼠肝脏线粒体氧化应激相关因子的影响,本实验将36只6周龄BALB/c小鼠随机分成4组,感染组经皮下接种200μL浓度为1×103TCID50/100μL PRV,对照组注射等量的PBS,分别于感染后48 h、72 h、96 h迫杀小鼠,采集新鲜的肝脏组织提取线粒体,通过ROS检测试剂盒检测各组小鼠线粒体活性氧(ROS),结果显示,与对照组相比,感染组小鼠肝脏线粒体ROS水平极显著增加(P<0.01)。采用分光光度法检测线粒体中的丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶体系(GSH-Px)和过氧化氢酶系统(CAT)变化,结果显示,与对照组相比,各感染组小鼠肝脏中线粒体MDA水平均显著增加(P<0.01),GSH水平显著降低(P<0.01)。采用线粒体膜电位MitoScreen检测试剂盒检测各组小鼠线粒体膜电位,结果显示感染组小鼠线粒体膜电位相较对照组出现了显著的去极化(P<0.01)。利用荧光定量PCR检测各组小鼠肝脏中氧化应激相关酶的基因转录水平,结果显示,感染组小鼠的SOD、GSH-Px和CAT 3种酶的基因转录水平相较于对照组均显著下调(P<0.01)。利用细胞凋亡检测试剂盒检测各组小鼠肝细胞凋亡情况,结果显示,与对照组比较,随着感染时间的延长,各感染组小鼠肝细胞凋亡比例显著或极显著增加(P<0.05,P<0.01)。本研究首次证实PRV感染小鼠后会导致肝脏线粒体出现氧化应激,从而造成肝细胞凋亡和线粒体膜电位降低,该结果为进一步研究PRV致病机理提供了一定的数据参考。

To study the effect of pseudorabies virus(PRV)infection on mitochondrial oxidative stress in liver,thirty-six 6-weekold BALB/c mice were randomly divided into 4 groups.Mice were injected subcutaneously with 200μL(1×103TCID50/100μL)PRV,and the control group was injected with the same amount of PBS.Liver tissues were collected to extract mitochondria at48 hours,72 hours and 96 hours post-infection,respectively.ROS production in all mice was detected by flow cytometry.The results showed that the level of mitochondrial ROS in liver in the infection group was significantly increased(P<0.01)compared with the control group.The levels of MDA,SOD,GSH,GSH-PX and CAT in mitochondria were detected by spectrophotometry.The results showed that MDA in the liver of each virus-infected mouse increased significantly(P<0.01).Compared with the control group,GSH content decreased significantly in all infected mouse(P<0.01).The transcriptional levels of SOD,GSH-Px and CAT were significantly down-regulated measured by real-time PCR(P<0.01).Furthermore,the apoptotic rate of mouse hepatocytes was detected by apoptosis detection kit,and the results showed that the proportion of apoptotic hepatocytes in each virus-infected mouse increased over time significantly or extremely significantly compared with the control group(P<0.05,P<0.01).In addition,the mitochondrial membrane potential was detected by MitoScreen kit,and the results showed that the mitochondrial membrane potential got significantly depolarized(P<0.01).In conclusion,this study confirmed for the first time that PRV infection could induce mitochondrial oxidative stress in liver and thus result in hepatocyte apoptosis as well as reduce mitochondrial membrane potential.The results provide data reference for further study on the pathogenesis of PRV.