Supplemental material

Cows with ketosis frequently display liver injury, driven primarily by increased influx of non-esterified fatty acids (NEFA) into the liver and subsequent heightened hepatic inflammatory responses. However, the underlying mechanisms driving these pathogenic events remain unclear. Neural precursor cell expressed developmentally downregulated 4 (NEDD4), a HECT-type E3 ubiquitin ligase, has emerged as a critical regulator of inflammatory signaling pathways and plays a pivotal role in the pathogenesis of liver diseases. In this study, we demonstrated a reduction in NEDD4 expression alongside evidence of inflammatory injury in the livers of cows with ketosis. Concurrently, receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was found to mediate NEFA-induced inflammatory injury in bovine hepatocytes. NEDD4 facilitates the ubiquitination and degradation of RIPK1 while inhibiting its phosphorylation, thereby alleviating NEFA-induced inflammatory cell injury. These findings suggest that targeting NEDD4 may represent a promising therapeutic strategy for mitigating liver injury in ketotic dairy cows.