The E3 ubiquitin ligase NEDD4 protects against nonesterified fatty acid–induced hepatic inflammatory injury in ketotic cows

Cows with ketosis frequently exhibit liver injury, primarily driven by increased influx of nonesterified fatty acids (NEFA) into the liver. This excess NEFA induces lipotoxicity, which triggers hepatic inflammatory responses and ultimately results in liver damage, further exacerbating ketosis. Neural precursor cell expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, plays a crucial role in regulating anti-inflammatory processes, yet its role in the hepatic injury in ketotic cows remains unclear. This study aimed to investigate the link between NEDD4 and hepatic inflammatory damage in ketotic cows. Liver biopsies and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 9 DIM. Primary bovine hepatocytes isolated from healthy one-day-old calves were treated with NEFA (0, 0.6, 1.2, or 2.4 mM, 12 h) or transfected with a bovine NEDD4 overexpression adenovirus or small interfering RNA targeting NEDD4 (48 h), followed by treatment with 1.2 mM NEFA (12 h). For degradation pathway experiment, Ad-NEDD4 transfected hepatocytes were exposed to chloroquine (50 µM, 6 h) or MG132 (10 µM, 10 h). For molecular cloning, hemagglutnin-tagged NEDD4 (HA-NEDD4) and flag-tagged bovine receptor-interacting protein kinase 1 (Flag-RIPK1) plasmids were expressed in 293T cells for 48 h, then treated with MG132 (10 µM, 10 h). Compared with the control group, ketotic cows showed higher serum concentrations of BHB, NEFA, tumor necrosis factor- α (TNF-α), and high mobility group box 1 (HMGB1), along with elevated activities of aspartate aminotransferase and alanine aminotransferase, but lower DMI, milk yield, and glucose. Histology analyses revealed inflammatory cell infiltration and pronounced necrosis. Hepatic IL-1β and the ratios of phosphorylated nuclear factor kappa B (p-NF-κB) to nuclear factor kappa B (NF-κB) and phosphorylated (p)-RIPK1 to RIPK1 were elevated. In vitro, the exogenous addition of 1.2 mM and 2.4 mM NEFA significantly reduced the abundance of NEDD4 in bovine hepatocytes, and increased the proportion of propidium iodide-positive hepatocytes. Concurrently, the concentrations of TNF-α and HMGB1 in the culture medium were elevated. Additionally, the protein expression levels of IL-1β and p-RIPK1 in bovine hepatocytes, as well as the ratio of p-NF-κB/NF-κB, were significantly increased. These findings indicate that NEFA can induce inflammatory injury in bovine hepatocytes in a dose-dependent manner. NEDD4 overexpression alleviated these NEFA-induced effects by reducing the RIPK1 protein expression and phosphorylation, whereas NEDD4 silencing exacerbated them. Co-immunoprecipitation demonstrated that NEDD4 interacted with RIPK1, promoting its ubiquitination and degradation of RIPK1 and suppressing phosphorylation. In summary, these data indicate that NEDD4 protects bovine hepatocytes from NEFA-induced inflammatory injury by regulating RIPK1 stability and activity, providing insights into its protective role in ketosis-related liver damage.