高NEFA血症可导致奶牛肝脂过度沉积，自噬体降解受阻，转录因子EB（TFEB）转录活性受损，但不清楚TFEB在高NEFA血症引发肝脂沉积过程中的作用机制。鉴于TFEB介导的自噬溶酶体通路通过多种途径调节脂质沉积，激活的mTOR通过磷酸化TFEB抑制其转录活性，本项目提出高浓度NEFA可通过过度激活mTOR抑制TFEB的转录活性，阻碍自噬溶酶体通路，引起肝脂沉积的科学假设。拟通过高NEFA血症奶牛的体内实验，结合体外培养奶牛肝细胞添加NEFA实验，并分别转染TFEB过表达腺病毒、TFEB-siRNA或添加mTOR抑制剂Rapa，检测mTOR信号通路、TFEB转录活性、自噬溶酶体信号通路和脂沉积状态，明确TFEB在高NEFA血症引起脂沉积过程中的作用，阐明mTOR-TFEB-自噬溶酶体通路调节脂沉积的作用机制，为防治以肝脂沉积为病理学特征的奶牛酮病和脂肪肝等糖脂代谢紊乱疾病提供理论基础。

High blood concentration of NEFA induces excessive hepatic lipid accumulation, blocks autophagy degradation and impairs transcription factor EB (TFEB) transcriptional activity in dairy cows. However, the role of TFEB in NEFA-induced hepatic lipid accumulation still obscure. TFEB-mediated autophagy-lysosome pathway regulates lipid accumulation by multi ways. Activated mTOR phosphorylates TFEB to inhibit its transcription activity. Besides, high concentration of NEFA displays lipotoxicity to liver. Accordingly, we hypothesize that high concentration of NEFA over-activated mTOR and subsequently inhibit the transcriptional activity of TFEB and block the autophagy-lysosome pathway, which further result in the hepatic lipid accumulation. In vivo experiment, we will collect blood and liver samples from healthy cows and dairy cows with high blood concentration of NEFA. In vitro experiment, we will isolate and culture bovine hepatocyte, treat with NEFA and transfect with TFEB overexpression adenovirus or TFEB-siRNA, or treat with mTOR inhibitor Rapa. Then, the mTOR signaling, TFEB transcriptional activity, autophagy and lysosomal signaling and lipid metabolism status will be evaluated. The above experiments aim to determine the role of TFEB in NEFA-induced hepatic lipid accumulation and reveal the mechanism of mTOR-TFEB-autophagy-lysosome pathway in regulation of hepatic lipid accumulation. These results will provide valuable information and functional target to precision prevention of hepatic lipid accumulation, which are induced by glycolipid metabolic disorder diseases, such as ketosis and fatty liver in dairy cow.