金属硫蛋白调节氧化应激下鸡胚肝脏能量代谢的作用机制

Oxidative stress during the embryonic stage induces the mitochondrial dysfunction of energy metabolism and then impairs the development of chick embryos. Metallothionein (MTs) as a key protein not only plays a great role in antioxidant ability, but also regulates mitochondrial energy metabolism against oxidative stress. Based on our previous findings, the increase of MT4 expressions induced by maternal zinc nutrition promoted the development of chick embryos. To identify the role and mechanism of MT4 in the regulation of hepatic mitochondrial energy metabolism to improve embryonic development subjected to oxidative stress, we firstly detect the distribution and localization of MT4 in hepatocyte of chick embryo subjected to oxidative stress. Then in vitro, the effect of MT4 agonist on the changes of mitochondrial energy metabolism in hepatocyte of chick embryo subjected to oxidative stress will be determined. Then, MT4 RNAi were applied in hepatocyte identify in the primary culture to investigate the signaling pathway of MT4 regulation on hepatic mitochondrial energy metabolism under oxidative stress. Finally, the effect of maternal dietary zinc supplementation on the hatchability, hepatic energy metabolism and expressions of MT4 and target genes of Akt/Nrf2/PPARα signaling pathway in chick embryos under oxidative stress was investigated. The outcomes from this project will reveal the mechanism of MT4 in the regulation of hepatic mitochondrial energy metabolism in chick embryos subjected to oxidative stress. It will be a great significance for how to improve embryonic development by maternal nutrition as targeting MT4 expression when embryos were subjected to oxidative stress.

家禽胚胎期氧化应激诱导线粒体能量代谢紊乱，是导致鸡胚发育异常的重要原因之一。金属硫蛋白（MTs）不仅发挥抗氧化作用，也很可能是介导线粒体能量代谢的关键蛋白。申请者前期研究发现，母代饲粮锌促鸡胚MT4表达改善胚胎发育。为深入研究MT4介导氧化应激下线粒体能量代谢调节鸡胚发育的机制，本项目以鸡胚肝脏为靶标，首先检测氧化应激下MT4在其定位与表达。随后，分别以离体鸡胚肝脏线粒体和原代培养鸡胚肝细胞为模型，探究MT4过表达对氧化应激下线粒体能量代谢的影响；同时结合基因沉默技术，阐明MT4调节氧化应激下线粒体能量代谢的信号通路与作用机制。最后，通过母代自然饲喂试验，运用胚蛋注射诱导氧化应激手段，研究母代锌营养促鸡胚MT4表达对其孵化性能和肝脏能量代谢的影响。研究结果不仅有望揭示MT4调节氧化应激下鸡胚肝脏能量代谢的作用机制，同时也为母代营养调控MT4靶点改善氧化应激下鸡胚发育提供试验依据。

家禽胚胎期氧化应激诱导线粒体能量代谢紊乱，是导致鸡胚发育异常的重要原因之一。金属硫蛋白很可能是介导线粒体能量代谢的关键蛋白之一。项目先后共研究了3部分内容，包括鸡胚肝脏线粒体氧化损伤模型的建立及其对能量代谢的影响、母代锌营养对氧化应激下鸡胚肝脏线粒体功能的调节作用及锌营养促MT4表达对氧化应激下线粒体能量代谢的调节机制研究。结果表明，通过胚蛋注射600μM/kg线粒体氧化剂t-BHP诱导鸡胚肝脏线粒体氧化应激模型，并发现其线粒体结构（ROS升高和膜电位降低）和能量代谢紊乱（氧化磷酸化水平下降，糖酵解增强），导致胚胎死亡率升高；接着，发现氧化应激条件下，母代锌营养可降低鸡胚肝脏线粒体ROS含量，提高胞浆和线粒体MT4含量及肝脏ATP含量，缓解鸡胚肝脏线粒体氧化应激损伤。然后，通过孵育100μM/t-BHP诱导原代细胞线粒体氧化应激模型，阐明锌促MT4表达可缓解氧化应激诱导的细胞能量代谢紊乱，与抑制PGC-1α/PPARγ通路的激活相关。最后，采用RNAi干扰手段诱导MT4表达抑制的原代细胞模型，验证了锌促MT4表达可介导PGC-1α/PPARγ通路调节线粒体能量代谢的信号机制。上述结果揭示了锌促MT4调节氧化应激下鸡胚肝脏能量代谢的作用机制，同时也为母代锌营养调控MT4靶点改善鸡胚发育提供试验依据。