β-Hydroxybutyrate inhibits apoptosis in bovine neutrophils through activating ERK1/2 and AKT signaling pathways

Ketosis in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation and high concentrations of blood β-hydroxybutyrate (BHB). Neutrophil apoptosis plays a key role in maintaining the balance of inflammation and functional capacity of circulating neutrophils in ketotic cows. The kinases ERK1/2 and AKT, as well as their downstream Bcl-2 family-mediated mitochondrial signaling, are important apoptosis-regulating pathways in neutrophils. The objective of our study was to investigate the effects of BHB on neutrophil apoptosis and the underlying regulatory mechanisms during ketosis. Neutrophils were isolated from 5 multiparous cows (within 3 wk postpartum) with serum BHB concentrations <0.6 mM and glucose concentrations >3.5 mM. In a series of experiments, neutrophils were treated with increasing concentrations of BHB (0, 0.6, 2, and 3 mM for 10 h) and time (0, 2, 4, 6, 8, and 10 h with 2 mM). Subsequently, a 2 mM BHB dose was used to challenge neutrophils for 8 h. Apoptosis rate of neutrophils and protein abundance of cleaved caspase 3 were lower after BHB treatment. Treatment with BHB decreased protein and mRNA abundance of the pro-apoptotic genes Bax (BAX) and Bad (BAD), whereas it increased mitochondrial membrane potential (MMP) and protein and mRNA of the anti-apoptotic genes Bcl-xL (BCL2L1) and Mcl-1 (MCL1). This indicated that a mitochondrial pathway was involved in the inhibition of neutrophil apoptosis via BHB. In addition, both SCH772984 (an inhibitor of the ERK1/2 signaling pathway) and MK-2206 (an inhibitor of the AKT signaling pathway) alleviated the BHB-induced anti-apoptotic function of the Bcl-2 family and the inhibition of MMP. Overall, our data demonstrated that high concentrations of BHB inhibit apoptosis in bovine neutrophils by activating the ERK1/2 and AKT signaling pathways. These findings provide a theoretical basis for the understanding of systemic inflammation in ketotic cows.