FOXA3 Alleviates Lipid Deposition in Primary Bovine Hepatocytes by Inhibiting SREBP1 and Cell Proliferation

During the periparturient period, negative energy balance (NEB) in dairy cows leads to increased concentrations of non-esterified fatty acids (NEFA) in the blood, which can induce fatty liver disease and ketosis. Forkhead box protein A3 (<i>FOXA3</i>) is a key transcription factor that regulates liver metabolism; however, its specific role in the pathogenesis of fatty liver in dairy cows remains unclear. This study aimed to investigate the mechanism by which <i>FOXA3</i> regulates hepatic lipid metabolism. We collected liver samples from dairy cows with fatty liver (<i>n</i> = 10) and from healthy cows (<i>n</i> = 10). Bovine primary hepatocytes were isolated from the liver tissue of calves (<i>n</i> = 5), followed by NEFA treatment, and we utilized <i>FOXA3</i> overexpression, immunofluorescence, and RNA sequencing (RNA-seq) to conduct our analysis. Our results demonstrated that <i>FOXA3</i> expression in the livers of cows with fatty liver was significantly lower than in healthy cows. NEFA treatment resulted in the downregulation of FOXA3 protein levels in hepatocytes, promoting triacylglycerol (TAG) accumulation and the expression of lipogenesis-related genes. Conversely, <i>FOXA3</i> overexpression mitigated NEFA-induced lipid accumulation, inhibited the expression of lipogenesis-related genes and proteins-particularly SREBP1-and affected cell proliferation, and the intracellular localization of <i>FOXA3</i> and <i>SREBP1</i>. RNA-seq analysis suggested that <i>FOXA3</i> may influence hepatic lipogenesis through pathways such as PI3K-Akt and the cell cycle. In summary, <i>FOXA3</i> mitigates NEFA-induced hepatic lipid accumulation through a dual mechanism: regulating <i>SREBP1</i> expression and inhibiting cellular proliferation. These findings highlight <i>FOXA3</i>'s potential as a novel target for the prevention and treatment of fatty liver disease in dairy cows.