锌指蛋白A20介导锌缓解鸡肠上皮细胞炎症反应的机制

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

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25μmol/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。

The research aimed to study the role of zinc and A 20 gene in relieving lipopolysaccharide(LPS)-induced inflammatory response in chicken intestinal epithelial cells by infecting chicken intestinal epithelial cells with zinc finger protein A 20 gene siRNA virus and A 20 gene adenovirus.The results showed that LPS significantly increased the gene expression levels of pro-inflammatory cytokines such as interleukin(IL)-1βand 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 25μmol/L zinc in vitro significantly alleviated LPS-induced higher gene expression levels and protein production of IL-8,IL-1βand 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 A 20 gene,the protein expression of A20 was significantly down-regulated(P<0.05),the gene expression levels of IL-1,IL-8 and TNF-αand their protein production were significantly increased(P<0.05),and the LPS-induced up-regulated gene expression levels of IL-8 and IL-1βand protein production of IL-1βand TNF-αwere also significantly enhanced(P<0.05).Compared with the control group,zinc addition was beneficial to the protein expression of A 20 and nuclear factor-κB(NF-κB)p 65,but inhibited protein expression of phospho-NF-κB p 65.However,no effects of zinc on the down-regulated protein expression of proteins A 20 and NF-κB p 65 and up-regulated protein expression of phospho-NF-κB p 65 and phospho-nuclear factor-κB inhibitory proteinα(IκBα)in cytoplasm,and up-regulated protein expression of phospho-NF-κB p65 in nucleus under A 20 gene silence were observed.Compared with the blank adenoviral group,infection with A 20 gene adenovirus extremely significantly increased the gene expression level of A 20(P<0.01),extremely significantly decreased the gene expression levels of IL-1βand 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-1βand significantly increased the gene expression level of NF-κB p 65(P<0.05).Therefore,A20-NF-κB p65 pathway plays an important role in the alleviation of LPS-induced inflammation by zinc.