PCV2感染对仔猪肝脏MyD88-NF-κB信号途径的影响

Effects of PCV2 infection on MyD88-NF-κB signal pathway in piglet’s liver

选取31头经检测猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)抗原和抗体均为阴性的5周龄断奶仔猪,随机分为对照组16头和试验组15头,对照组仔猪每头滴鼻4 mL PBS,试验组仔猪每头滴鼻4 mL 5×105TCID50.mL-1PCV2悬液。于PCV2接种当天剖杀4头仔猪作为对照组,分别于14、21和35 d剖杀4头对照组和5头试验组仔猪,采集肝脏。用激光共聚焦显微镜检测核因子κB/P65(NF-κB/P65)蛋白的核易位变化;蛋白提取法分别提取肝脏细胞核蛋白和胞浆蛋白,Western blot定量检测细胞核中NF-κB/P65和细胞浆中p-IκBα、MyD88蛋白含量的变化;电泳迁移率(EMSA)法检测细胞核中NF-κB DNA的结合率变化。检测结果显示,PCV2接种仔猪,肝脏中NF-κB/P65蛋白核易位逐渐增多,到21 d达到峰值;p-IκBα、MyD88、NF-κB/P65 DNA结合率在接种后均先升高后趋于恢复,并于21 d达到高峰。与对照组仔猪相比,肝脏中MyD88和NF-κB/P65蛋白含量以及NF-κB DNA结合率在14和21 d试验组均显著升高(P<0.05),35 d含量变化不显著;21 d时试验组p-IκBα蛋白含量显著升高。相关性分析显示,NF-κB/P65蛋白含量与MyD88含量、NF-κB DNA结合率之间存在显著正相关,相关系数分别是0.566和0.528。结果表明,PCV2可通过激活MyD88启动NF-κB信号途径;通过IκBα的磷酸化降解激活NF-κB,并促进其进行核易位,使NF-κB与DNA发生结合,调控相关炎性细胞因子的转录和表达。

Thirty one piglets which were free of porcine circovirus type 2（PCV2）and procine reproductive and respiratory syndrome virus（PRRSV）antibody together with antigen were chosen as the experiment animals,and randomly distributed in two groups：the control group（n=16）and the experimental group（n=15）.Every piglet in the experimental group was inoculated with PCV2,and the livers were collected on 0 day-post-infected（DPI）from 4 piglets of the control group.On 14,21 and 35 DPI,the livers were collected from 4 piglets of the control group and 5 piglets of the experimental group.Nuclear factor kappa B/P65（NF-κB/P65）translocation in livers was detected with confocal laser scanning microscope.The proteins were extracted from livers,and then the protein level of P65 in nucleus,phosphorylation of inhibitory kappa B（p-IκBα）and myeloid differentiation factor 88（MyD88）in cytoplasm were detected by Western blot,and NF-κB DNA binding rate was detected by electrophoretic mobility shift assay（EMSA）.The results showed that NF-κB/P65 translocation increased gradually in time-dependent manner and reached a peak on 21 DPI,and that p-IκBα,MyD88,NF-κB DNA binding rate increased gradually with time,and reached a peak on 21 DPI.The MyD88,NF-κB/P65,NF-κB DNA binding rate significantly increased（P0.05）on 14 and 21 DPI,compared with control group.But they were unconspicuous on 35 DPI.The phosphorylation-IκBα of the experimental group was significantly higher than that of the control group on 21 DPI.The results of correlation analysis showed NF-κB/P65 had significant positive correlation with MyD88 and NF-κB DNA binding rate,the P-value were 0.022 and 0.036 respectively,and correlation coefficients were 0.566 and 0.528 respectively.The results demonstrated that PCV2 can activate NF-κB signal pathway by MyD88.PCV2 can activate NF-κB pathway through degradation of IκBα phosphorylation and lead to translocation of NF-κB,increase NF-κB DNA binding rate,and regulate assistance gene expression.