Nuclear Transcription Factor-&kappa;B/&beta;-Catenin Signaling Pathway Mediates Enterotoxigenic Escherichia coli Causing IPEC-J2 Cell Injury

DOU Caixia; LI Haihua; SUN Zeyang; WANG Qian; SHANG Zhiyuan; QIAO Jiayun

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

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

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

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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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

Cite this article

DOU Caixia , LI Haihua , SUN Zeyang , WANG Qian , SHANG Zhiyuan , QIAO Jiayun . Nuclear Transcription Factor-κB/β-Catenin Signaling Pathway Mediates Enterotoxigenic Escherichia coli Causing IPEC-J2 Cell Injury[J]. Chinese Journal of Animal Nutrition, 2020 , 32(12) : 5893 -5902 . DOI: 10.3969/j.issn.1006-267x.2020.12.042

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