Effects Of STAT3 And Its Negative Regulatory Protein SOCS3 On Expression Of Inflammatory Cytokine IL-6 Induced By CSFV

Classical swine fever(CSF),caused by Classical swine fever virus(CSFV),is a critical infectious disease in pigs.In recent years,the epidemic characteristics of CSF has been changed a lot and vaccine failure has occurred.Therefore,it is necessary to investigate the mechanism underlying pathogenesis of CSFV and to develop the new strategy to control the CSF.To understand such molecular mechanism,we performed in vitro and in vivo experiments.We examined the mRNA and protein levels of IL-6,SOCS3 and p-STAT3 in pigs and PK-15 cells infected with CSFV Shimen strain.RT-PCR and qRT-PCR assays were used to detect the mRNA levels of IL-6 and SOCS3 of various tissues of SPF experimental pigs,which were infected with CSFV Shimen strain for 5 days.The phosphorylation levels of STAT3 protein were analyzed by Western blotting.In vitro,we determined the mRNA expression levels of IL-6 and SOCS3 in PK-15 cells infected with CSFV Shimen strain for 5 different time points.The phosphorylation levels of STAT3 protein in vitro were also investigated by Western blotting.In addition,we successfully constructed PK-15 cell lines that stably interfere with the expression of IL-6Rα,STAT3 and SOCS3 respectively.We detected that knockdown of IL-6Rα had effect on the phosphorylation levels of STAT3 in PK-15 cells after the CSFV Shimen strain infection.Later,PK-15 cells expressing shSOCS3 and shSTAT3 were infected with CSFV Shimen strain to detect the effect of STAT3 and SOCS3 on the IL-6.We also explored its effect on IL-6 expression by overexpression or constitutive activation of STAT3.The results are presented as follows:1.The expression of IL-6,SOCS3 and p-STAT3 was obviously increased in the spleens,mesenteric lymph nodes,inguinal lymph nodes,mandibular lymph nodes and tonsil of SPF pigs after the CSFV Shimen strain infection.Indicating that the CSFV infection can induce the IL-6 expression,activate the JAK-STAT3 signaling pathway and the negative regulatory protein SOCS3 of the cytokine signaling pathway in vivo;2.The expression of IL-6,SOCS3 and p-STAT3 in PK-15 cells was up-regulated after the CSFVShimen strain infection;all are time-dependent increase first and then decline.This indicates that the CSFV infection can induce the IL-6 expression,activate the STAT3 and the negative regulatory protein SOCS3 of the cytokine signaling pathway in vitro;3.The expression of p-STAT3 in PK-15 cell lines with sh-IL-6Rαwas lower than that of the control group after the CSFV Shimen strain infection,indicatingthat the CSFV infection can activate the IL-6/IL-6Rα-mediated the phosphorylation of STAT3;4.Down regulation of STAT3 also caused less expression of p-STAT3 in PK-15 cell line as compared to the control group after the CSFV infection,and the expression level of IL-6 was higher than that in thecontrol group.The overexpression of STAT3 wild type and constitutively activated STAT3 in PK-15 cells infected with CSFV leads to a lower level of IL-6 than that in the control group.These results indicate that the activation stat of STAT3 is negatively correlated to the level of IL-6 during the CSFV infection;5.PK-15 cells expressing lower levels of SOCS3 showed high expression of p-STAT3 in comparison to control,and the expression level of IL-6 was lower than that in the control group.Indicating that the SOCS3 induced by CSFV may negatively regulate the STAT3 activation and affect the expression level of IL-6;The studies above reveal that the CSFV infection can induce the IL-6-mediated JAK/STAT3 signaling pathway.The SOCS3 induced by CSFV may negatively regulate the STAT3 activation and there is a positive correlation with IL-6.These resultswill provide a new theoretical basis for the further study on the pathogenic mechanism of CSFV.