谷氨酰胺对呕吐毒素诱导IPEC-J2细胞凋亡和炎症的影响

Effects of glutamine on deoxynivalenol induced apoptosis and inflammation of IPEC-J2 cells

[目的]本试验以猪空肠上皮细胞(IPEC-J2)为模型,探讨谷氨酰胺(Gln)对呕吐毒素(DON)诱导的IPEC-J2细胞凋亡和炎症的影响。[方法]通过MTT方法测定细胞活力来选择适宜的Gln浓度,以不添加Gln和DON的细胞为空白对照组,试验组分别为0.75 mmol·L^-1 Gln组,2.0μg·mL^-1 DON组和2.0μg·mL^-1 DON+0.75 mmol·L^-1 Gln组,处理24 h后测定各组细胞凋亡率、活性氧自由基(ROS)及细胞凋亡和炎症相关基因和蛋白的表达水平。[结果]与对照组相比,DON处理24 h细胞凋亡比例和ROS含量(ROS荧光密度/细胞总数)显著升高(P<0.01);与DON组相比,DON+Gln组显著降低细胞的凋亡比例和ROS含量(P<0.01)。与对照组相比,DON组上调IPEC-J2细胞白细胞介素1β(IL^-1β)、白细胞介素6(IL-6)、环氧合酶2(COX-2)及肿瘤坏死因子α(TNF-α)炎症相关基因和Caspase-3及Caspase-8凋亡相关基因的表达水平;与DON组相比,DON+Gln组下调IPEC-J2细胞IL^-1β、COX-2、Caspase-3、Caspase-8、BAK和Bcl-2基因的表达水平。蛋白免疫荧光结果显示,与对照组相比,DON组上调IPEC-J2细胞Caspase-3,核转录因子κB(NF-κB)和磷酸化核转录因子κB(phospho-NF-κB)蛋白的表达,而添加Gln后(DON+Gln组)下调了相关蛋白的表达。[结论]Gln通过清除DON诱导的IPEC-J2细胞中过量的ROS及调节炎症和凋亡相关基因及蛋白的表达量来缓解由DON引起的肠道上皮细胞损伤。

[Objectives]In this study,intestinal porcine epithelial cells(IPEC-J2 cells)were used as a model to investigate the effects of glutamine(Gln)on deoxynivalenol(DON)induced apoptosis and inflammation of IPEC-J2 cells.[Methods]The appropriate Gln concentration was selected by MTT cell viability assay,and the cells without Gln and DON were used as the control group.The treated groups were 0.75 mmol·L^-1 Gln,2.0μg·mL^-1 DON and 2.0μg·mL^-1 DON+0.75 mmol·L^-1 Gln,respectively.After 24 h,the apoptosis,reactive oxygen species(ROS),some genes and proteins associated with apoptosis and inflammation were measured.[Results]Compared with the control group,the percentage of apoptosis and ROS content(ROS fluorescence density/cell total)significantly increased after DON treatment for 24 h(P<0.01).Compared with the DON group,DON+Gln significantly decreased the percentage of apoptosis and ROS content(P<0.01).Compared with the control group,DON up-regulated the expression of genes involved in inflammation and apoptosis,such as interleukin-1β(IL^-1β),interleukin-6(IL-6),cyclooxygenase 2(COX-2),tumor necrosis factor alpha(TNF-α),Caspase-3,Caspase-8;DON+Gln down-regulated the expression of IL^-1β,COX-2,Caspase-3,Caspase-8,BAK and Bcl-2 genes compared with DON group.Protein immunofluorescence revealed that compared with the control group,DON up-regulated the expression of Caspase-3,nuclear factor kappa B(NF-κB)and phosphorylated nuclear factor kappa B(phospho-NF-κB)protein,and Gln down-regulate the expression of related proteins.[Conclusions]Gln alleviated the damage of IPEC-J2 cells caused by DON by decreasing ROS production and regulating the expression of genes and protein involved in inflammation and apoptosis.