β-Hydroxybutyrate Exacerbates Klebsiella pneumoniae -Induced Inflammatory Injury and Milk Synthesis Impairment via the GPR109A/NF-κB Signaling Axis

Abstract Ketotic cows exhibit a heightened mastitis risk through an unknown mechanism. This study investigated the influence of β-hydroxybutyrate (BHBA) on mastitis development. Mammary tissues from ketotic cows with mastitis showed more severe pathological damage and suppression of lactation-related genes. BHBA pretreatment further increased NF-κB pathway activation, facilitated expressions of pro-inflammatory cytokines, and suppressed lactation proteins in Klebsiella pneumoniae (K. pneumoniae)-infected BMECs, thereby exacerbating inflammatory injury and milk synthesis impairment. Molecular docking, DARTS, and CETSA assays confirmed that BHBA directly bound to GPR109A. GPR109A knockdown blocked the activation of the NF-κB pathway, alleviated inflammatory injury, and mitigated milk synthesis impairment. Meanwhile, similar findings were identified with PTX (an inhibitor of GPR109A) treatment. These findings elucidated the role of BHBA and its endogenous receptor GPR109A in K. pneumoniae-induced inflammatory injury and milk synthesis impairment, which provided valuable insight for understanding the interplay between metabolic imbalance and postpartum mammary health.