Hepatocyte necroptosis is associated with liver damage in dairy cows with ketosis

Dairy cows with ketosis frequently exhibit impaired liver function and inflammation. Necroptosis, a form of programmed cell death associated with cellular lysis, releases damage-associated molecular patterns (DAMP) into injured tissues, thereby amplifying inflammatory responses and tissue damage. However, the role of necroptosis in the liver of ketotic cows remains unknown. The present study aimed to investigate whether necroptosis is associated with hepatic damage in dairy cows with ketosis. Dairy cows were selected as either the clinically healthy control group (n = 15) or a ketotic group diagnosed with ketosis, and were matched for lactation numbers (median = 3, range = 2-4) and DIM (median = 6 d, range = 3-9 d). Liver tissue samples were collected via percutaneous needle biopsy, and blood samples were obtained by coccygeal venipuncture. Primary bovine hepatocytes were isolated from the liver of 1-d-old calves. In vivo experiments showed significant increases in the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), and glutamate dehydrogenase (GLDH) in the blood of ketotic cows. Concurrently, the mRNA abundance of IL-6 (IL6), tumor necrosis factor-α (TNF), and IL-1β (IL1B) in the liver of ketotic cows also significantly increased. Histopathological examination revealed significant cytoplasmic vacuolation, lytic necrosis, and inflammatory cell infiltration in the liver of ketotic cows, along with increased CD11b immunofluorescence intensity. Furthermore, the expression levels of necroptosis-related proteins, including the ratio of phosphorylated receptor-interacting protein kinase 1 (p-RIPK1)/receptor-interacting protein kinase 1 (RIPK1), phosphorylated receptor-interacting protein kinase 3 (p-RIPK3)/receptor-interacting protein kinase 3 (RIPK3), and phosphorylated mixed lineage kinase domain-like pseudokinase (p-MLKL)/mixed lineage kinase domain-like pseudokinase (MLKL) significantly increased in the liver of ketotic cows. The mRNA abundance of RIPK1, RIPK3, and MLKL also significantly increased. Immunohistochemical analysis confirmed elevated p-MLKL and p-RIPK3 expression in the liver of ketotic cows. In vitro experiments showed that compared with the control group, bovine hepatocytes treated with 10 ng/mL TNF-α significantly activated necroptosis signaling, as ascertained by an increase in ratios of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL, and in the mRNA expression of RIPK1, RIPK3, and MLKL. Moreover, TNF-α treatment significantly upregulated the mRNA abundance of inflammatory cytokines IL1B and IL6, as well as the activity of ALT, AST, GGT, and GLDH in the culture medium of hepatocytes. Importantly, treatment with 100 µM Necrostatin 1 or 3 µM GSK-872 notably attenuated TNF-α-induced necroptosis signaling, inflammatory cytokine expression, and the activity of ALT, AST, GGT, and GLDH. In conclusion, these data suggest that necroptosis is associated with hepatic damage, and may be a potential therapeutic target to ameliorate liver dysfunction in dairy cows with ketosis.