丁酸梭菌介导mTOR信号通路调控猪肠上皮细胞炎症反应的分子机制

The molecular mechanism of Clostridium butyricum mediated mTOR signaling pathway regulating the inflammatory response of porcine intestinal epithelial cells

本试验旨在研究丁酸梭菌(CB)介导雷帕霉素靶蛋白(mTOR)信号通路调控产肠毒素大肠杆菌K88(ETEC K88)感染猪肠上皮细胞(IPEC-J2)炎症反应的分子机制。按试验步骤处理细胞,利用1×10^(3) cfu/mL的ETEC K88感染IPEC-J2以及400 nmol/L的mTOR抑制剂处理IPEC-J2,到达作用时间后,分别收集细胞及培养上清,然后按照猪肿瘤坏死因子(TNF-α)和猪白细胞介素-8(IL-8)检测试剂盒说明书进行细胞因子检测,以及使用荧光定量PCR(qPCR)法进行ZO-1、occludin和mTOR表达量的检测。结果表明:与ETEC组相比,抑制剂+ETEC组、CB+ETEC组和抑制剂+CB+ETEC组ZO-1和occludin的表达量均显著升高,或有升高趋势,ZO-1的表达量分别升高了86.59%、31.48%和133.98%,occludin的表达量分别升高了69.34%、18.63%和87.52%,而mTOR的表达量分别降低了40.81%、11.62%和52.43%。与CB+ETEC组相比,抑制剂+CB+ETEC组在mTOR活性被抑制后ZO-1和occludin的表达量极显著升高了77.97%和58.07%,而mTOR的表达量极显著降低了46.18%,CB与抑制剂协同逆转ETEC造成的紧密连接蛋白表达下降。综上所述,ETEC K88能够激活mTOR信号通路,而CB通过介导mTOR信号通路能够降低ETEC K88感染IPEC-J2引起的炎症反应,并提高紧密连接蛋白的表达量,从而减轻细胞损伤。

This study was aimed to investigate the molecular mechanism of Clostridium butyricum(CB)mediated mammalian target of rapamycin(mTOR)signaling pathway regulating the inflammatory response of porcine intestinal epithelial cells(IPEC-J2)infected with enterotoxigenic Escherichia coli K88(ETEC K88).IPEC-J2 were processed according to the steps,using the 1×10^(3) cfu/mL ETEC K88 infection IPEC-J2 and 400 nmol/L mTOR inhibitor treatment IPEC-J2.The cells and culture supernatant were collected when reach the action time,respectively.Then cytokines TNF-αand IL-8 were detected according to the kit instruction,as well as using qPCR method to detect the expression quantity of ZO-1,occludin and mTOR.The results showed that:compared with the ETEC group,the expression levels of ZO-1 and occludin in inhibitor+ETEC group,CB+ETEC group and inhibitor+CB+ETEC group were significantly increased(P<0.05,P<0.01),or there was a rising trend.The expression levels of ZO-1 was increased by 86.59%,31.48%and 133.98%,respectively,the expression of occludin was increased by 69.34%,18.63%and 87.52%,respectively,while the expression of mTOR was decreased by 40.81%,11.62%and 52.43%,respectively.Compared with CB+ETEC group,the expression levels of ZO-1 and occludin in inhibitor+CB+ETEC group were significantly increased(P<0.01)by 77.97%and 58.07%after mTOR activity was inhibited,while the expression level of mTOR was significantly decreased by 46.18%in inhibitor+CB+ETEC group.CB and inhibitor synergism reversed the decrease of tight junction protein expression caused by ETEC K88.In conclusion,ETEC K88 could activate the mTOR signaling pathway,but CB could reduce the inflammatory response caused by ETEC K88,and increase the expression level of tight junction protein by mediating mTOR signaling pathway,thus reducing cell damage.