亮氨酸对早期断奶仔猪肝中内质网应激的影响

Effect of leucine on endoplasmic reticulum stress induced by early weaning in the liver of piglets

为探究日粮中亮氨酸水平对早期断奶仔猪(长×大)肝内质网应激的影响,利用透射电镜、细胞核质分离和蛋白质免疫印迹等技术,研究了在亮氨酸处理下肝细胞形态和内质网应激特征蛋白质的表达规律及对早期断奶诱导的肝内质网应激的影响。结果显示:在早期断奶诱导内质网应激模型中,与正常不断奶组相比,14日龄早期断奶组仔猪肝细胞中内质网口径增大,结构紊乱,同时细胞核内内质网应激相关蛋白XBP1的表达水平显著上调(P<0.05);在体外Hep G2细胞培养模拟试验中,培养基中葡萄糖和亮氨酸的缺失都能显著上调细胞核内内质网应激相关蛋白ATF4和ATF6α剪切体蛋白的表达(P<0.05),而在培养基中重补亮氨酸后,核内定位的ATF4蛋白出现了进一步的上调(P<0.05)。不同亮氨酸水平日粮的饲喂试验结果显示,在21日龄未断奶条件下,日粮亮氨酸水平对ATF4和ATF6α剪切体在细胞核内的表达无显著影响(P>0.05),而断奶24 h后,2.10%亮氨酸组仔猪肝细胞核内的ATF4蛋白表达水平显著高于1.66%亮氨酸组(P<0.05)。上述结果表明,早期断奶能够诱导仔猪肝内质网应激的发生,而亮氨酸对早期断奶引发的肝内质网应激具有重要的调节功能。

To investigate the effects of dietary leucine supplementation on endoplasmic reticulum（ER） stress in the liver of early-weaning piglets, we explored the morphology of liver cells, expression pattern of ER stress-associated proteins, and effects on ER stress induced by early weaning under leucine treatment using transmission electron microscopy（TEM）, nucleo-cytoplasmic fractionation and immunoblotting assays. The results showed that in the early weaning-induced ER stress model, the diameter of ER in the liver cells derived from 14-day-old early-weaning piglets was increased compared with the normal group, while the structure of ER was disordered, and the expression level of ER stress-associated protein XBP1 in the nucleus of liver cells was significantly up-regulated（P〈0.05）. In the Hep G2 cells cultured in vitro, the ER stress-associated proteins ATF4 and cleaved ATF6α were significantly upregulated（P〈0.05） with the absence of either glucose or leucine in the medium, while after re-stimulation with leucine,ATF4 expression was further up-regulated in the nucleus of Hep G2（P〈0.05）. By feeding weaning piglets with diets containing different levels of leucine, we found that dietary leucine levels had no significant effect on the expression of ATF4 and cleaved ATF6α in the nucleus of liver cells derived from 21-day-old non-weaning piglets（P〈0.05）. After weaning 24 h, the expression of ATF4 in the nucleus of liver cells derived from piglets fed with diet containing 2.10%leucine was significantly higher than that of 1.66% leucine group（P〈0.05）. In summary, early weaning can induce ER stress in the liver cells of piglets, and leucine plays crucial roles in regulating ER stress induced by early weaning.