动物营养学报 >

2020 , Vol. 32 >Issue 12: 5893 - 5902

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.12.042

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

核转录因子-κB/β-连环蛋白信号通路介导肠产毒性大肠杆菌引致IPEC-J2细胞损伤

  • 窦彩霞 ,
  • 李海花 ,
  • 孙泽阳 ,
  • 王倩 ,
  • 尚智援 ,
  • 乔家运
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  • 1. 天津师范大学生命科学学院天津市动植物抗性重点实验室, 天津 300387;
    2. 天津农学院动物科学与动物医学学院, 天津市农业动物繁育与健康养殖重点实验室, 天津 300384
窦彩霞(1995-),女,山西大同人,硕士研究生,研究方向为动物营养与饲料。E-mail:1490191412@qq.com

收稿日期: 2020-05-13

  网络出版日期: 2020-12-07

基金资助

天津市自然科学基金项目（18JCYBJC30000）；天津市自然科学基金项目"Wnt/β-catenin信号通路介导嗜酸乳杆菌调节仔猪肠黏膜上皮屏障功能的分子机制"；天津市"131"创新型人才团队（20180338）；天津市高校"学科领军人才培养计划"

收起

Nuclear Transcription Factor-κB/β-Catenin Signaling Pathway Mediates Enterotoxigenic Escherichia coli Causing IPEC-J2 Cell Injury

  • DOU Caixia ,
  • LI Haihua ,
  • SUN Zeyang ,
  • WANG Qian ,
  • SHANG Zhiyuan ,
  • QIAO Jiayun
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  • 1. Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University, Tianjin 300387, China;
    2. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China

Received date: 2020-05-13

  Online published: 2020-12-07

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摘要

本试验旨在探讨肠产毒性大肠杆菌（ETEC）造成IPEC-J2细胞损伤的分子机制。ETEC K88感染、核转录因子-κB（NF-κB）抑制剂处理IPEC-J2细胞以及小干扰RNA（siRNA）沉默IPEC-J2细胞中β-连环蛋白（β-catenin）基因的表达后，采用定量PCR（qPCR）法检测细胞中NF-κB、闭锁小带蛋白-1（ZO-1）、闭锁蛋白（occludin）、Toll样受体2（TLR2）、Toll样受体4（TLR4）、β-连环蛋白（β-catenin）、细胞周期蛋白D1（cyclin D1）和人髓细胞增生原癌基因（c-Myc）的mRNA表达量，用Western blotting法检测细胞中ZO-1、occludin的蛋白表达量和p65 NF-κB的磷酸化水平，用酶联免疫吸附测定（ELISA）法检测细胞培养上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-8（IL-8）含量和乳酸脱氢酶（LDH）活性。结果表明：ETEC K88作用细胞6或24 h时，ZO-1和occludin的蛋白表达量以及β-catenin和c-Myc的mRNA表达量均极显著降低（P<0.01），TNF-α、IL-8含量和LDH活性显著上升（P<0.05），p65 NF-κB的磷酸化水平在ETEC K88作用细胞1、3和6 h时均极显著上升（P<0.01），TLR2、TLR4的mRNA表达量在ETEC K88作用细胞6和12 h时均极显著上升（P<0.01）。NF-κB被抑制后，TNF-α、IL-8和LDH的活性均极显著降低（P<0.01），ZO-1和occludin的蛋白表达量以及NF-κB的mRNA表达量均极显著升高（P<0.01）。siRNA沉默β-catenin基因后，各时间点β-catenin、cyclin D1和c-Myc的mRNA表达量均极显著降低（P<0.01），LDH的活性显著上升（P<0.05）。综上所述，ETEC K88通过上调NF-κB信号通路引起炎症反应，下调β-catenin信号通路活化抑制IPEC-J2细胞的增殖，从而诱导IPEC-J2细胞损伤。

关键词： ETEC K88; IPEC-J2细胞; NF-&kappa; B信号通路; &beta; -catenin信号通路

本文引用格式

窦彩霞 , 李海花 , 孙泽阳 , 王倩 , 尚智援 , 乔家运 . 核转录因子-κB/β-连环蛋白信号通路介导肠产毒性大肠杆菌引致IPEC-J2细胞损伤[J]. 动物营养学报, 2020 , 32(12) : 5893 -5902 . DOI: 10.3969/j.issn.1006-267x.2020.12.042

Abstract

This study aimed to explore the molecular mechanism of enterotoxigenic Escherichia coli (ETEC) causing damage to IPEC-J2 cells. After ETEC K88 infection and nuclear transcription factor-κB (NF-κB) inhibitor treatment of IPEC-J2 cells and small interfering RNA (siRNA) silencing of β-catenin gene expression in IPEC-J2 cells, quantify PCR (qPCR) method was then performed to determine the mRNA expression levels of NF-κB, zonula occludens-1 (ZO-1), occludin, Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), β-catenin, cyclin D1 and myelocytomatosis oncogene (c-Myc) in cells, Western blotting method was conducted to determine the protein expression levels of ZO-1 and occludin as well as the phosphorylation level of p65 NF-κB in cells, and enzyme-linked immunosorbent assay (ELISA) method was used to determine the tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) contents and lactate dehydrogenase (LDH) activity in cell culture supernatant. The results showed as follows: either 6 or 24 h ETEC K88 infection could enable the protein expression levels of ZO-1, occludin and the mRNA expression levels of β-catenin and c-Myc to extremely significantly decline (P<0.01), and the contents of TNF-α, IL-8 and LDH activity to significantly rise (P<0.05); the phosphorylation level of p65 NF-κB was extremely significantly at 1, 3 and 6 h post ETEC infection (P<0.01); plus, the mRNA expression levels of TLR2 and TLR4 were extremely significantly increased with 6 or 12 h ETEC infection (P<0.01). After the successful inhibition of NF-κB, the contents of TNF-α, IL-8 and LDH activity were extremely significantly decreased (P<0.01), while the protein expression levels of ZO-1 and occludin and the mRNA expression level of NF-κB were extremely significantly increased (P<0.01). When the β-catenin gene was silenced by small interference RNA (siRNA), the mRNA expression levels of β-catenin, cyclin D1 and c-Myc were extremely significantly decreased (P<0.01), and the LDH activity was significantly improved at each time point (P<0.05). To sum up, ETEC K88 is able to generate inflammatory response by up-regulating NF-κB signaling pathway accompanied by down-regulating the activation of β-catenin signaling pathway. In this case, the proliferation of IPEC-J2 cells is inhibited, thereby inducing IPEC-J2 cells injury.

Key words： ETEC K88; IPEC-J2 cells; NF-κB signaling pathway; β-catenin signaling pathway

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