Abstract
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.
Highlights
Ketosis is a metabolic disease characterized by increased blood concentration of ketone bodies, which typically occurs in dairy cows soon after calving
We found linear (P < 0.0001) and quadratic effects (P < 0.0001) for apoptosis rate due to the increase in dose of blood β-hydroxybutyrate (BHB) (Figure 1 A, B)
Compared with control (0 mM BHB), no difference in apoptosis rate was detected with 0.6 mM BHB (P = 0.706, Figure 1 B)
Summary
Ketosis is a metabolic disease characterized by increased blood concentration of ketone bodies, which typically occurs in dairy cows soon after calving. Ketotic cows are characterized by high concentrations of nonesterified fatty acids and BHB in the blood, along with systemic inflammation (Itle et al, 2015; Shen et al, 2021). The latter was associated with higher odds of developing other diseases, such as displaced abomasum, metritis, mastitis, lameness, and gastrointestinal disorders (Suthar et al, 2013; Berge and Vertenten, 2014; Pryce et al, 2016). Neutrophils contribute to the early innate immune response by rapidly migrating into inflamed tissues, and killing and clearing pathogens, along with amplifying inflammation (Daha, 2011) These cells are characterized by a short lifespan, with “old” or activated neutrophils eventually undergoing apoptosis as a safe elimination process mediated via programmed cellular death (Savill, 2000). Apoptotic neutrophils are cleared by macrophages, a process that helps control inflammation by inhibiting
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