沙门菌通过NF-κB/β-catenin信号通路引致IPEC-J2损伤的分子机制

doi:10.11843/j.issn.0366-6964.2021.025

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (1): 235-245.doi: 10.11843/j.issn.0366-6964.2021.025

沙门菌通过NF-κB/β-catenin信号通路引致IPEC-J2损伤的分子机制

王怡梦¹, 刘雪姣¹, 王倩², 魏庆¹, 窦彩霞², 尚智援², 乔家运², 李海花^1*

1. 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;
2. 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

收稿日期:2020-06-05 出版日期:2021-01-23 发布日期:2021-01-19
通讯作者: 李海花,主要从事动物营养与免疫研究,E-mail:lihaihua@tjau.edu.cn
作者简介:王怡梦(1993-),女,河南周口人,硕士生,主要从事微生物与免疫学研究,E-mail:wymeng0811@126.com
基金资助:
国家自然科学基金（31702147）；天津市高校“中青年骨干创新人才培养计划”；天津市高校“学科领军人才培养计划”；天津市“131”创新型人才团队（20180338）；天津市自然科学基金（20JCZDJC00190）

Molecular Mechanism of the Injury of IPEC-J2 Caused by Salmonella via NF-κB/β-catenin Signaling Pathway

WANG Yimeng¹, LIU Xuejiao¹, WANG Qian², WEI Qing¹, DOU Caixia², SHANG Zhiyuan², QIAO Jiayun², LI Haihua^1*

1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Breeding, College of Animal Science and Animal Medicine, Tianjin Agricultural University, Tianjin 300384, China;
2. Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China

Received:2020-06-05 Online:2021-01-23 Published:2021-01-19

摘要/Abstract

摘要： 为研究沙门菌引致IPEC-J2损伤的分子机制，试验用沙门菌刺激IPEC-J2，在特定时间收集细胞及其培养上清液，采用荧光定量PCR或ELISA检测细胞紧密连接蛋白、炎症反应相关蛋白和细胞增殖相关蛋白的表达以及乳酸脱氢酶（LDH）含量，以确定该沙门菌能够引起IPEC-J2损伤，并进一步采用NF-κB抑制剂和小干扰RNA预处理IPEC-J2，分析了NF-κB/β-catenin信号通路在沙门菌引起IPEC-J2损伤中的调控作用。结果显示，与对照组相比，沙门菌感染组紧密连接蛋白ZO-1和Occludin mRNA表达量在感染后6和24 h呈极显著下降（P<0.01）；促炎性细胞因子TNF-α和IL-8的生成量以及LDH释放量在感染后6、12和24 h呈极显著上升（P<0.01），NF-κB p65的mRNA表达在感染后1、3和6 h也呈极显著上升（P<0.01）；细胞增殖蛋白cyclin D1和c-Myc的mRNA表达在感染后6、12和24 h呈显著下降（P<0.05），其转录调控因子β-catenin的mRNA表达在感染后24 h呈极显著下降（P<0.01）。与沙门菌感染组相比，抑制剂+沙门菌感染组ZO-1和Occludin的mRNA表达量在12和18 h呈极显著增高（P<0.01），而NF-κB p65、TNF-α和IL-8的表达以及LDH释放量在各时间点均呈极显著降低（P<0.01）；siRNA-β-catenin+沙门菌处理组的β-catenin、cyclin D1和c-Myc的表达量在各时间点均呈极显著降低（P<0.01），LDH释放量在6和12 h均呈极显著增加（P<0.01），但在18 h增加不显著（P>0.05）。本试验结果表明，沙门菌激活了NF-κB信号通路，促进了促炎性细胞因子的生成，加重了细胞损伤；同时，沙门菌抑制了β-catenin信号通路，降低了细胞增殖蛋白和紧密连接蛋白的表达，影响了细胞修复，从而出现细胞损伤与细胞修复之间的失调，最终呈现出细胞损伤。本试验从NF-κB/β-catenin信号通路的角度揭示了沙门菌引致IPEC-J2损伤的分子机制。

关键词: 沙门菌, IPEC-J2, NF-κB信号通路, β-catenin信号通路

Abstract: In order to study the molecular mechanism of IPEC-J2 injury induced by Salmonella, we stimulated IPEC-J2 with Salmonella and collected the cells and culture supernatant at pointed times. The expression of tight junction proteins, inflammation-related proteins, cell proliferation-related proteins and the content of lactate dehydrogenase (LDH) were detected by fluorescence quantitative PCR or ELISA to confirm that Salmonella could cause IPEC-J2 damage. We also further explored the regulation of NF-κB/β-catenin signaling pathway in Salmonella induced-IPEC-J2 injury using NF-κB inhibitors and small interference RNA. The results showed that, compared with the control group, the mRNA expression levels of tight junction proteins ZO-1 and Occludin in the Salmonella-infected group were extremely significantly decreased at 6 and 24 h after infection (P<0.01); the production of pro-inflammatory cytokines TNF-α, IL-8, and the release of LDH, were extremely significantly increased at 6, 12 and 24 h after infection (P<0.01), and the mRNA expression of NF-κB p65 was also extremely significantly increased at 1, 3 and 6 h after infection (P<0.01); and the mRNA expression of cell proliferation proteins cyclin D1 and c-Myc significantly decreased at 6, 12 and 24 h after infection (P<0.05), and the mRNA expression of transcription regulatory factor β-catenin was extremely significantly decreased at 24 h after infection (P<0.01). Compared with Salmonella-infected group, the mRNA expression levels of ZO-1 and Occludin in the inhibitor+Salmonella-infected group were extremely significantly increased at 12 and 18 h (P<0.01), while the levels of NF-κB p65, TNF-α, IL-8 and LDH content were extremely significantly decreased at all time points (P<0.01); the levels of β-catenin, cyclin D1 and c-Myc were extremely significantly decreased at each time point in siRNA-β-catenin+Salmonella treatment group (P<0.01), the release of LDH extremely significantly increased at 6 and 12 h (P<0.01), but no significant increase at 18 h after infection (P>0.05). The results showed that Salmonella activated the NF-κB signaling pathway, promoted the production of pro-inflammatory cytokines, and aggravated the IPEC-J2 damage; meanwhile, Salmonella inhibited the β-catenin signaling pathway, decreased the expression of cell proliferation proteins and tight junction proteins, and affected the cell repair, which leads to the imbalance between cell damage and cell repair, and finally presents cell damage. This study revealed the molecular mechanism of IPEC-J2 damage caused by Salmonella from the perspective of NF-κB/β-catenin signaling pathway.

Key words: Salmonella, IPEC-J2, NF-κB signaling pathway, β-catenin signaling pathway

中图分类号:

S852.61⁺2

王怡梦, 刘雪姣, 王倩, 魏庆, 窦彩霞, 尚智援, 乔家运, 李海花. 沙门菌通过NF-κB/β-catenin信号通路引致IPEC-J2损伤的分子机制[J]. 畜牧兽医学报, 2021, 52(1): 235-245.

WANG Yimeng, LIU Xuejiao, WANG Qian, WEI Qing, DOU Caixia, SHANG Zhiyuan, QIAO Jiayun, LI Haihua. Molecular Mechanism of the Injury of IPEC-J2 Caused by Salmonella via NF-κB/β-catenin Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 235-245.

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沙门菌通过NF-κB/β-catenin信号通路引致IPEC-J2损伤的分子机制

Molecular Mechanism of the Injury of IPEC-J2 Caused by Salmonella via NF-κB/β-catenin Signaling Pathway

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