小反刍兽疫病毒通过TLR2/MyD88/NF-κB信号通路介导LGD-2细胞炎症反应

Inflammatory response in peste des petits ruminants virus infected LDG-2 cells is mediated by TLR2/MyD88/NF-κB signaling pathway

采用MTT试验、间接免疫荧光试验(IFA)和实时荧光定量PCR(qRT-PCR)观察小反刍兽疫病毒(peste des petits ruminants virus,PPRV)在山羊肾细胞(LDG-2)的增殖,采用Western blot和qRT-PCR观察PPRV感染对TLR2、MyD88、NF-κB信号通路相关因子及其下游炎症因子表达水平的影响。结果显示,PPRV感染后36 h,细胞存活率显著降低并产生明显细胞病变,PPRV-N基因mRNA表达水平显著上调;PPRV感染细胞中TLR2、MyD88、p-p65、p-IκBα表达水平、p-p65/p65和p-IκBα/IκBα比值以及下游炎症因子TNFα、IL-1β、IL-4和IL-10的mRNA表达水平均显著升高。用C29阻断PPRV诱导的TLR2激活,PPRV感染细胞中PPRV-N的mRNA、TLR2、MyD88、p-p65、p-IκBα表达水平及p-p65/p65和p-IκBα/IκBα比值、下游炎症因子IL-1β和IL-4的mRNA表达水平均显著降低。结果表明,PPRV感染可激活TLR2/MyD88/NF-κBα信号通路,促进病毒的复制和扩散;阻断PPRV诱导的TLR2激活,可抑制MyD88/NF-κB信号通路和病毒复制。

MTT assay,indirect immunofluorescence assay(IFA)and real-time fluorescence quantitative PCR(qRT-PCR)were used to observe the proliferation of PPRV in goat kidney cells(LDG-2).Western blot and qRT-PCR were used to evaluate the effects of PPRV infection on the expression levels of TLR^(2),MyD88,NF-κB signaling pathway-related factors and their downstream inflammatory factors.The results indicated that the significantly decreased cell survival rate and obvious cytopathic effect were observed at 36 h post PPRV infection,and the mRNA expression level of PPRV-N gene was significantly up-regulated.At the same time,the expression levels of TLR^(2),MyD88,p-p65 and p-IκBα,the ratio of p-p65/p65 and p-IκBα/IκBαand the mRNA expression levels of downstream inflammatory factors TNFα,IL-1β,IL-4 and IL-10 were significantly increased.Moreover,the expression levels of PPRV-N mRNA,TLR^(2),MyD88,p-p65 and p-IκBα,the ratio of p-p65/p65 and p-IκBα/IκBαand the mRNA expression levels of downstream inflammatory factors IL-1βand IL-4 in PPRV-infected cells were significantly decreased in the presence of the inhibitor C29 of TLR^(2).These findings implied that the TLR^(2)/MyD88/NF-κBαsignaling pathway can be activated by PPRV infection,which is beneficial to the replication and spread of the virus.Blocking down the activation of TLR^(2)can inhibit MyD88/NF-κB signaling pathway and viral replication in PPRV-infected cells.