The Nutritional Regulation Of Endoplasmic Reticulum Stress Induced By Early Weaning Stress In Liver Of Piglets

Early weaning has been proved to be a critical technology in the pig production. However, early weaning may lead to physiological and psychological impacts on piglets, and then lead to early weaning stress. Endoplasmic reticulum(ER) is a critical site of protein processing and Ca2+storage in cell. It is sensitive to stress. Various stressors can induce ER stress in cells and tissues. ER stress is critical for injury resistance and adaptability. It remains to be further studied that whether early weaning will induce ER stress in the liver of piglets and the role of ER stress in the early weaning stress.This project composed of three parts:1. Whether early-weaning stress will induce ER stress in piglets;2. Exploring the mechanism of ER stress under nutrition deprivation in HepG2cell;3. Feeding piglets with diet contained different levels of leucine to explore the role of leucine in regulating ER stress in early weanig piglets.Below are the results in this study:1. Compared with control (unweaned piglets), Transmission electron micrographs of endoplasmic reticulum morphology in livers derived from early-weaning piglets of Large White showed that, endoplasmic reticulum dilation was observed in weaned piglets at15d (the first day after weaning). It was noted that situation deteriorated in weaned piglets at16d. Given that XBP1is involved in the pathway of unfold protein response induced by endoplasmic reticulum stress, its expression in hepatocyte nucleus was detected. Related results demonstrated that, in comparison with unweaned piglets, XBP1expression in hepatocyte nucleus increased significantly in early-weaning piglets at15d (P<0.05). Those evidences indicated that the early weaning stress induce the endoplasimic reticulum stress in the liver of piglets.2. HepG2cells were incubated with EBSS-contained, low glucose-contained, glucose-deficient, leucine-deficient or amino acid-deficient medium respectively. We detected the expression of protein related to endoplasmic reticulum stress in HepG2cells by Western blot. The expression of ATF4increased significantly in the nuclear of HepG2cells when exposed to EBSS-contained medium. Treating the cells with low glucose-contained medium led to the increase of ATF4expression in the cytoplasm and nuclear of HepG2cells (P﹤0.05). When the cells exposed to glucose-deficient, the expression of ATF4, ATF6α and CHOP increased markedly in the cytoplasm (P<0.05), and the expression of ATF4in the nuclear also increased (P<0.05). The expression of CHOP in the cytoplasm and the ATF4, ATF6a in the nuclear increased notablely in leucine-deprivation HepG2cells (P<0.05), and the expression of XBP1in the cytoplasm decreased (P<0.05). Incubating the cells with amino acid-deficient medium, the levels of CHOP in cytoplasm and ATF4in nuclear rose markedly (P<0.05). And our results suggest that the expression of endoplasmic reticulum stress related proteins, ATF4(in the cytoplasm), ATF6a (in the nuclear) and XBP1(in the nuclear) increased when leucine defects for2hour (P﹤0.01). Moreover the increase was lower at4hour than at2hour (P>0.05).3. Determination of ATF4and ATF6a expression in HepG2cells resupplied with leucine (the final concentration is10mM) after leucine starvation by Western blot. Comparing with the group of leucin-deprivation, The expression of ATF4and ATF6a decreased (P<0.05). This result indicates that the endoplasmic reticulum stress was eased. Drawing on Western blot, we detected the expression of ATF4associated with endoplasmic reticulum stress in livers of piglets whose diets contain various concentrations of leucine. It is found that ATF4expression in hepatocyte nucleus decreased significantly in weaned piglets fed with2.1%and2.44%leucine (P<0.05), illuminating that leucine exerted regulation effect on hepatocyte endoplasmic reticulum stress induced by early weaning in piglets.Taken together, early weaning stress can induce endoplasmic reticulum stress in the liver of the piglets; The diet of early-weaning piglet which the leucine level is2.1%and2.44%respectively can easing the endoplasmic reticulum stress in the liver of piglet.