Supplemental material

Dairy cows with ketosis often experience liver dysfunction, characterized by hepatocellular injury and increased hepatic inflammation response. However, the underlying mechanism of these pathogenic events remains unclear. Necroptosis, a form of programmed cell death that promotes organ inflammatory injury, plays a critical role in the pathogenesis of liver diseases. In this study, we confirmed the occurrence of necroptosis in the liver of dairy cows with ketosis. We found that tumor necrosis factor-alpha (TNF-α), as a ketosis-associated inflammatory cytokine, induced necroptosis in bovine hepatocytes. Furthermore, inhibiting necroptosis via the RIPK1 kinase inhibitor Nec-1 and RIPK3 kinase inhibitor GSK-872 alleviated TNF-α-induced cellular damage. These findings suggest that targeting necroptosis could be a potential therapeutic strategy to mitigate hepatic damage in dairy cows with ketosis.