核黄素介导CHOP信号通路调控鸭胚肝细胞凋亡的分子机制

核黄素是家禽胚胎正常发育过程中所必需的营养素。申请人前期系列研究发现，种母鸭核黄素缺乏导致后代胚胎发育受阻，并诱导鸭胚肝脏发生内质网应激和细胞凋亡，但其具体分子机制尚不清楚。基于CHOP是内质网应激诱导细胞凋亡的关键通路，本研究提出“核黄素通过内质网应激CHOP信号通路调控鸭胚肝细胞凋亡”的科学假设。本项目拟先采用鸭胚原代肝细胞模型，探明核黄素对CHOP信号通路、内质网应激及细胞凋亡的影响；再采用CHOP基因沉默和过表达技术，通过研究内质网应激蛋白、CHOP信号通路及其下游靶蛋白和凋亡蛋白的mRNA和蛋白表达水平的变化，在细胞水平解析CHOP信号通路在核黄素抗凋亡过程中的关键作用及其机制；最后，通过母代自然饲喂，结合胚蛋注射抑制剂以阻断CHOP信号手段，从体内最终证实核黄素介导CHOP信号通路调控鸭胚肝细胞凋亡的分子机制。为种禽生产中合理使用核黄素以提高繁殖性能、改善胚胎发育提供理论依据。

Riboflavin is an essential nutrient in the embryonic development in poultry. Our previous series of studies showed that maternal riboflavin deficiency in ducks caused abnormal embryonic development of offspring, as well as endoplasmic reticulum stress and hepatocyte apoptosis in embryo liver. However, the underlying molecular mechanisms remain unclear. It is shown that CHOP is the key signaling pathway in the process of endoplasmic reticulum stress induces apoptosis. Thus we put forward a scientific hypothesis that “riboflavin modulates hepatocyte apoptosis of duck embryos by endoplasmic reticulum stress CHOP signaling pathway”. In the present project, therefore, we will first use primary cultured hepatocytes of duck embryos model to investigate the effects of riboflavin on CHOP signaling pathway, endoplasmic reticulum stress, and hepatocyte apoptosis. Secondly, we will use CHOP gene-silencing and the gene-overexpression techniques, and determine the mRNA and protein expression of proteins in endoplasmic reticulum stress, CHOP and its downstream target proteins, and apoptosis, to elucidate the key roles of CHOP signaling pathway in the process of riboflavin resists hepatocyte apoptosis and the underlying mechanisms in vitro. At last, a vivo experiment with naturally feeding duck breeders, in combination with injecting the inhibitor into embryos to suppress CHOP signaling pathway, will be conducted to further verify the molecular mechanism that riboflavin deficiency induces apoptosis mediated with CHOP signaling pathway in vivo. The outcomes from the present project will provide theoretical support for improving reproductive performance and promoting embryonic development by riboflavin supplementation properly in the diet of breeding poultry.