撒坝猪源大肠埃希氏菌HPI对泛素蛋白酶体介导的TLR4/NF-κB信号通路关键因子表达的影响

Effects of UPS Mediated E.coli HPI from Saba Piglets on Expressions of Key Factors in TLR4/NF-κB Signaling Pathway

为探讨撒坝猪源大肠埃希氏菌(E.coli)的耶尔森强毒力岛(HPI)对泛素蛋白酶体(UPS)介导的TLR4/NF-κB信号通路关键因子的影响,将猪小肠上皮细胞(IPEC-J2)分为5组,空白对照组、HPI阳性株感染组、HPI阴性株感染组、HPI阳性株+MG-132组、HPI阴性株+MG-132组。于试验后的第4、6、12、24 h收集各组细胞,用试剂盒检测泛素蛋白酶体活性,荧光定量PCR法检测TLR4、MyD88、NF-κB和IκB-α的mRNA水平,ELISA法检测NF-κB、IκB-α及炎性因子IL-1和TNF-α含量。结果显示,IPEC-J2细胞感染E.coli后,与空白对照组相比,感染组中泛素蛋白酶体活性水平、TLR4、MyD88、NF-κB和IκB-α的mRNA水平及细胞炎性因子IL-1和TNF-α的蛋白含量总体上调,且HPI阳性株感染组均高于HPI阴性株感染组;用E.coli HPI感染经MG-132处理后的IPEC-J2细胞,泛素蛋白酶体活性下降,TLR4/NF-κB信号通路中关键因子的转录水平及炎性因子的蛋白含量下降,且低于空白对照组。试验结果表明撒坝猪源E.coli HPI能激活泛素蛋白酶体介导的TLR4/NF-κB信号通路,可通过上调TLR4、MyD88、NF-κB和IκB-α的mRNA水平,促进炎性因子IL-1和TNF-α的释放,诱发炎症反应。

To investigate the effect of UPS mediated E.coli HPI from Saba piglets on the key factors of TLR4/NF-κB signaling pathway,IPEC-J2 cells were divided into 5 groups:blank control group,HPI positive group,HPI negative group,HPI positive+MG-132 group and HPI negative+MG-132 group.The cells of each group were collected at the 4,6,12 and 24 hours after the test,the activities of UPS were detected by kit,detection of mRNA levels of TLR4,MyD88,NF-κB and IκB-αby real-time PCR,and detect expression levels of NF-κB,IκB-α,IL-1 and TNF-αwith ELISA.The results showed that IPEC-J2 cells were infected with E.coli,compared with the blank control group,the levels of UPS activity,TLR4,MyD88,NF-κB and IκB-αmRNA,and the protein contents of IL-1 and TNF-αin the infected group were all up-regulated,and the HPI positive group was higher than the HPI negative group;When E.coli HPI was used to infect IPEC-J2 cells treated with MG-132,the activity of UPS decreased,and the transcriptional level of key factors in TLR4/NF-κB signaling pathway and the protein content of inflammatory factors showed a general downward trend,which was lower than that in blank control group.The results indicated that E.coli HPI from Saba pig could activate TLR4/NF-κB signaling pathway mediated by UPS,which could up-regulate the transcription levels of TLR4,MyD88,NF-κB and IκB-αand promote the release of IL-1 and TNF-αto enhance inflammatory response.