SIRT4调控奶牛乳腺上皮细胞热应激损伤的分子机制研究

热应激诱导乳腺上皮细胞损伤，是夏季奶牛泌乳降低的重要诱因，但损伤机制尚未阐明。前期研究发现，热应激显著降低去乙酰化酶4（Sirtuin4，SIRT4）的表达，且该差异表达与线粒体功能障碍密切相关。因此推测，热应激下SIRT4参与调控维持线粒体功能，减少细胞损伤，阻抑泌乳降低。本项目拟：①明确热应激下奶牛乳腺组织SIRT4的差异表达与泌乳降低、线粒体功能障碍的必然联系；②体外培养乳腺上皮细胞，建立敲除和过表达SIRT4的细胞模型，探究SIRT4对泌乳和线粒体功能的调控作用；③抑制AMPK-mTOR信号通路，检测线粒体途径凋亡和自噬；同时利用蛋白质谱和免疫共沉淀技术，建立SIRT4与AMPK-mTOR信号通路的联系，最终阐明SIRT4调控奶牛乳腺上皮细胞热应激损伤的分子机制。本项目预期结果可为研究缓解奶牛热应激的分子干预技术提供理论支撑。

Heat stress-induced dairy cow mammary epithelial cells (DCMEC) injury is a critical cause of the reduced lactation in summer, but the mechanism of heat stress-induced DCMEC injury has not been elucidated. Our previous works showed that heat stress significantly reduced the expression of Sirtuin 4(SIRT4) in DCMEC, which was closely related to mitochondria dysfunction. Therefore, we speculated that SIRT4 could maintain mitochondrial function, attenuate heat stress-induced DCMEC injury and the decrease of lactation. Therefore, we carry out the following studies: Firstly, we investigate the relationship between SIRT4 expression level and heat stress-induced the decrease of lactation and mitochondrial dysfunction in mammary gland tissues. Secondly, the SIRT4 knockout and overexpressed DCMEC are cultured in vitro and are used to determine the effect of SIRT4 on mitochondrial function and lactation-related genes expression. Thirdly, inhibition of AMPK-mTOR signaling pathway to investigate its effect on mitochondrial apoptosis and autophagy. In addition, protein mass spectrometry and Co-IP technologies are used to investigate the regulatory mechanism of SIRT4 in AMPK-mTOR signaling pathway, which will clarify the regulatory mechanism of SIRT4 in heat stress-induced DCMEC injury. This project expects to provide a theoretical support for the study of molecular technology to relieve heat stress-induced damage in dairy cows.