基于“p53-溶酶体-线粒体”途径探讨脂联素修复鸡骨骼肌细胞氧化损伤机制

Oxidative damage of skeletal muscle seriously reduces chicken meat quality. The studies indicated that p53 activation could trigger lysosome and mitochondrial damage, leading to cell oxidative damage and apoptosis. Previously, our research demonstrated that adiponectin has been found resisting oxidative stress in skeletal muscle. But whether this feature is dependent on p53-mediated mitochondrial and lysosomal damage pathway is unclear. This project will investigate the mechanism of adiponectin repairs poultry skeletal muscle cell oxidative damage through "p53-lysosome-mitochondria" pathway. The main contents are as follows: 1) Oxidative damage model of poultry skeletal muscle cell will be constructed. Adiponectin will be used to treat model cells, cells are harvested for the time series analysis of p53 expression and for the detection of membrane permeability of mitochondria and lysosomes. 2) In order to reveal the interactions of p53, lysosomes and mitochondria in the process of anti-oxidation damage by adiponectin, we respectively use the agonists and inhibitors of p53, mitochondria and lysosomal to treat the model cells together with adiponectin, laser confocal and CoIP for observing protein localization. Finally, by revealing the molecular mechanism of how adiponectin block oxidative damage in poultry skeletal muscle cell, this project aims to provide evidence to support the usage of adiponectin in releasing oxidative stress.

骨骼肌氧化损伤严重降低鸡肉品质。研究发现，p53蛋白活化能触发溶酶体和线粒体损伤，导致细胞氧化损伤甚至凋亡。本课题组前期研究结果证实，脂联素具有修复骨骼肌氧化损伤的功能，但这种功能是否依赖于p53介导的溶酶体和线粒体损伤途径尚不清楚。本项目通过对“p53-溶酶体-线粒体”代谢途径的研究，探讨脂联素修复鸡骨骼肌细胞氧化损伤机理。主要研究内容如下：1）使用H2O2处理鸡骨骼肌细胞构建氧化应激损伤模型，脂联素处理模型细胞，时序分析p53表达、溶酶体及线粒体损伤的变化；2）脂联素处理模型细胞后，分别用p53、溶酶体、线粒体的激动剂和抑制剂处理细胞，激光共聚焦观察蛋白定位结合Co-IP技术研究脂联素修复氧化损伤过程中p53、溶酶体、线粒体三者之间的互作机制。通过以上研究，阐明脂联素修复鸡骨骼肌氧化损伤的关键途径，为应用脂联素修复氧化应激损伤提供理论依据。

本项目分别探讨了脂联素对模型细胞C2C12以及鸡骨骼肌细胞氧化损伤的修复机制。.（一）探讨了脂联素对小鼠骨骼肌细胞C2C12氧化损伤的保护作用及分子机制。脂联素预处理骨骼肌细胞C2C12之后，用过氧化氢处理来模拟氧化损伤。结果显示，脂联素对小鼠骨骼肌细胞氧化损伤具有细胞保护作用，脂联素缓解过氧化氢诱导的生长抑制，增加细胞抗氧化能力，清除过氧化氢诱导的活性氧的能力。脂联素抑制过氧化氢诱导的自噬流增加，抑制Pink1、Parkin的增加，抑制线粒体自噬，部分抑制溶酶体和线粒体的共定位。此外，脂联素抑制过氧化氢诱导的细胞凋亡，抑制Bax的转录水平和蛋白水平及凋亡相关蛋白的转录水平。这些结果表明，脂联素在氧化应激诱导的线粒体自噬过程中具有一定的调节作用，抑制细胞凋亡。由于其抗氧化损伤的作用，可以考虑将其作为一个骨骼肌氧化损伤有关疾病的防治药物。.（二）构建鸡骨骼肌细胞氧化应激模型，探讨脂联素对肉鸡骨骼肌细胞氧化损伤的修复机制，为肉鸡抗应激高效养殖提供依据。分离培养肉鸡骨骼肌卫星细胞并鉴定，过氧化氢处理构建肉鸡骨骼肌氧化应激模型，p53抑制剂PFT-α与脂联素共同处理氧化应激模型细胞，研究p53参与脂联素修复氧化应激的机制。结果显示：脂联素可通过抑制p53的表达，改善细胞溶酶体和线粒体膜通透性、恢复膜电位、抑制凋亡基因和蛋白的表达、以及细胞凋亡相关信号途径，修复肉鸡骨骼肌细胞的氧化损伤。