肠道病毒71型激活人横纹肌肉瘤细胞中JNK1/2信号转导通路

Enterovirus 71 activates the JNK1/2 signaling pathway in human rhabdosarcoma cells

目的探讨c-Jun N端激酶(JNK)信号转导通路在肠道病毒71型(EV71)感染中的作用。方法台盼蓝染色法检测不同浓度抑制剂SP600125对人横纹肌肉瘤细胞(RD)活性的影响;实时荧光定量PCR及western blot检测EV71感染RD细胞后VP1 mRNA及蛋白质的表达水平;western blot检测JNK1/2、c-Fos、c-Jun蛋白及其磷酸化水平,并分析JNK1/2抑制剂SP600125对EV71复制及JNK1/2信号通路的影响。结果台盼蓝染色结果表明,与空白对照组比较,5和10μmol/L实验组存活率差异无统计学意义(P均>0.05),而20μmol/L抑制剂组细胞存活率明显降低(P<0.05);实时荧光定量PCR及western blot结果表明,与对照组相比,实验组在EV71感染RD细胞8 h后,其VP1 mRNA及蛋白质的表达水平均明显下降(P均<0.01)。此外,western blot检测结果证实EV71感染RD细胞后,其JNK1/2、c-Fos和c-Jun的蛋白质磷酸化水平均明显升高(P均<0.05)。而经抑制剂SP600125处理可明显下调其JNK1/2、c-Fos、c-Jun磷酸化水平(P均<0.05)。结论 JNK1/2信号通路可被EV71感染有效激活,并与EV71复制有关。

Objective To investigate the role of c-Jun N-terminal kinase（ JNK1/2） signaling pathway in enterovirus 71（ EV71） infection. Methods The effects of different concentrations of SP600125 on the activity of human rhabdosarcoma（ RD） cells were detected by trypanbalu staining. The levels of VP1 mRNA and protein in EV71-infected RD cells were detected by real time Q-PCR and western blot,respectively. The levels of total and phosphorylated JNK1/2,c-Fos and c-Jun protein were determined by western blot.Last,the effects of JNK1/2 inhibitor SP600125 on EV71 replication and JNK1/2 signaling pathway were analyzed. Results The results of trypanbalu staining showed that 5 and 10 μmol/L of SP600125 didn＇t influence on the activity of RD cells（ P〈0. 05）,while20 μmol/L of SP600125 decreased the survival of RD cells significantly（ P〈0. 05）. Compared with the control,the expression levels of VP1 mRNA and protein in EV71-infected RD cells decreased obviously at 8 hours post-infection（ P〈0. 01）. In addition,after RD cells were infected EV71,the levels of phosphorylated JNK1/2,c-Fos and c-Jun increased significantly（ P〈0. 05）. However,the pretreatment of SP600125 decreased the phosphorylation levels of JNK1/2,c-Fos and c-Jun protein obviously（ P〈0. 05）. Conclusion EV71 infection may effectively activate the JNK1/2 signaling pathway in RD cells,which may be related to EV71 replication.