Lipopolysaccharide-induced glycolytic reprogramming drives H3K18 lactylation-mediated inflammatory injury in buffalo mammary epithelial cells

Buffalo mastitis impairs milk production and quality, yet the underlying epigenetic mechanisms remain unclear. While LPS-induced glycolytic activation and lactate accumulation contribute to inflammation, their role in histone lactylation-mediated injury is unexplored. Our results revealed that mammary tissues from buffalo with clinical mastitis exhibited elevated LPS levels, upregulation of inflammatory markers, enhanced apoptosis, and markedly higher levels of lactate, Pankla, and H3K18la compared to healthy controls. Similarly, an LPS-induced mouse mastitis model recapitulated the inflammatory and apoptotic features observed in buffalo tissues, confirming LPS as a key driver of mammary tissue injury. In vitro, LPS treatment of buffalo mammary epithelial cells (BuMECs) upregulated proinflammatory and proapoptotic markers while downregulating antiapoptotic genes. RNA-seq and western blot analyses further revealed that LPS enhanced the expression and activity of key glycolytic enzymes, such as HK2, PKM2, PDK1, and LDHA, thereby promoting glycolytic flux, increasing lactate production, and elevating Pankla and H3K18la levels. CUT&Tag analysis suggested that H3K18la may facilitate the recruitment of transcriptional regulators, such as DDIT3, NF-κB1, and CEBPD, potentially linking lactylation to the activation of inflammatory and apoptotic pathways. Importantly, inhibition of HK2 reduced lactate accumulation, decreased H3K18la levels, and substantially attenuated inflammatory injury in BuMECs. Together, these findings identify the HK2-lactate-H3K18la axis as a central mechanism in LPS-induced inflammatory injury in BuMECs and underscore its potential as a therapeutic target for alleviating mastitis and enhancing mammary health in dairy buffalo.