动物营养学报 >

2019 , Vol. 31 >Issue 5: 2254 - 2266

DOI: https://doi.org/10.3969/j.issn.1006-267x.2019.05.032

分子营养 Molecular Nutrition

锌指蛋白A20介导锌缓解鸡肠上皮细胞炎症反应的机制

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  • 中国农业大学动物科技学院, 动物营养学国家重点实验室, 北京 100193
高金鑫(1993-),女,山东青岛人,硕士研究生,从事家禽营养与饲料科学研究。E-mail:gaojinxin93@126.com

收稿日期: 2018-10-22

  网络出版日期: 2019-05-15

基金资助

国家自然科学基金项目（30928018）

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Mechanism of Zinc Relieves Inflammatory Response Mediated by Zinc Finger Protein A20 in Chicken Intestinal Epithelial Cells

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  • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Received date: 2018-10-22

  Online published: 2019-05-15

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摘要

本研究通过对鸡肠上皮细胞感染锌指蛋白A20基因siRNA病毒和A20基因腺病毒，探究锌与A20基因对缓解脂多糖（LPS）诱导的鸡肠上皮细胞炎症反应的作用。结果表明：LPS可显著上调鸡肠上皮细胞促炎因子白细胞介素（IL）-1β和IL-8的基因表达水平（P<0.05），作用的最佳剂量和时间分别是10 ng/mL和6 h。体外添加25 μmol/L锌可显著缓解LPS诱导的鸡肠上皮细胞IL-8、IL-1β和肿瘤坏死因子-α（TNF-α）基因表达水平和蛋白产量的提高以及Toll样受体4（TLR4）的转录激活。肠上皮细胞感染A20基因siRNA病毒后，A20的蛋白产量显著下调（P<0.05），IL-1β、IL-8和TNF-α的基因表达水平和蛋白产量显著升高（P<0.05），同时加剧LPS诱导的IL-8、IL-1β基因表达水平和IL-1β、TNF-α蛋白产量的提高（P<0.05）。相对于对照组，加锌可促进胞浆中A20和核因子-κB（NF-κB） p65的蛋白表达，并下调磷酸化NF-κB p65的蛋白表达；在进行A20基因沉默后，导致胞浆中A20、NF-κB p65蛋白表达下调和磷酸化NF-κB p65、磷酸化核因子κB抑制蛋白α（IκBα）蛋白表达上调以及胞核中磷酸化NF-κB p65的蛋白表达升高，而加锌并不能改善。与感染空白腺病毒组相比，肠上皮细胞感染A20腺病毒可极显著提高A20的基因表达水平（P<0.01），并极显著降低IL-1β、IL-8的基因表达水平（P<0.01），且可极显著降低添加LPS导致的IL-1β、IL-8和TLR4基因表达水平的上升（P<0.01）。锌添加可显著降低IL-1β的基因表达水平，显著提高NF-κB p65的基因表达水平（P<0.05）。综上所述，锌可通过A20-NF-κB p65信号通路缓解LPS诱导的鸡肠上皮细胞炎症反应。

关键词： 肉鸡; 原代肠上皮细胞; 锌; A20; siRNA; 腺病毒

本文引用格式

高金鑫, 李昌武, 张贝贝, 郭双双, 袁建敏, 聂伟, 呙于明 . 锌指蛋白A20介导锌缓解鸡肠上皮细胞炎症反应的机制[J]. 动物营养学报, 2019 , 31(5) : 2254 -2266 . DOI: 10.3969/j.issn.1006-267x.2019.05.032

Abstract

The research aimed to study the role of zinc and A20 gene in relieving lipopolysaccharide(LPS)-induced inflammatory response in chicken intestinal epithelial cells by infecting chicken intestinal epithelial cells with zinc finger protein A20 gene siRNA virus and A20 gene adenovirus. The results showed that LPS significantly increased the gene expression levels of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-8 in chicken intestinal epithelial cells (P<0.05), and the optimal dose and time for LPS administration were 10 ng/mL and 6 h, respectively. Addition of 25 μmol/L zinc in vitro significantly alleviated LPS-induced higher gene expression levels and protein production of IL-8, IL-1β and tumor necrosis factor-α (TNF-α) in chicken intestinal epithelial cells, as well as the transcriptional activation of toll-like receptor 4 (TLR4) (P<0.05). After intestinal epithelial cells were infected with the siRNA virus of A20 gene, the protein expression of A20 was significantly down-regulated (P<0.05), the gene expression levels of IL-1, IL-8 and TNF-α and their protein production were significantly increased (P<0.05), and the LPS-induced up-regulated gene expression levels of IL-8 and IL-1β and protein production of IL-1β and TNF-α were also significantly enhanced (P<0.05). Compared with the control group, zinc addition was beneficial to the protein expression of A20 and nuclear factor-κB (NF-κB) p65, but inhibited protein expression of phospho-NF-κB p65. However, no effects of zinc on the down-regulated protein expression of proteins A20 and NF-κB p65 and up-regulated protein expression of phospho-NF-κB p65 and phospho-nuclear factor-κB inhibitory protein α (IκBα) in cytoplasm, and up-regulated protein expression of phospho-NF-κB p65 in nucleus under A20 gene silence were observed. Compared with the blank adenoviral group, infection with A20 gene adenovirus extremely significantly increased the gene expression level of A20 (P<0.01), extremely significantly decreased the gene expression levels of IL-1β and IL-8 (P<0.01), and the increasing gene expression levels of IL-1β, IL-8 and TLR4 induced by LPS were extremely significantly alleviated (P<0.01). Zinc addition significantly reduced the gene expression level of IL-1β and significantly increased the gene expression level of NF-κB p65(P<0.05). Therefore, A20-NF-κB p65 pathway plays an important role in the alleviation of LPS-induced inflammation by zinc.

Key words： broiler; primary intestine epithelial cells; zinc; A20; siRNA; adenovirus

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