Free Fatty Acids and Endotoxins Synergically Induce Pyroptosis in Bovine Hepatocytes

<b>Background/Objectives</b>: Elevated circulating non-esterified fatty acids (NEFAs) are closely associated with hepatic inflammatory injury in dairy cattle, simultaneously with the entry of lipopolysaccharide (LPS) into the liver. This study aimed to investigate the synergistic effects of NEFAs and LPS on pyroptosis in bovine hepatocytes. <b>Methods</b>: Primary bovine hepatocytes were allocated into control, NEFA, NEFA + LPS, NEFA + LPS + Caspase-1 inhibitor, and NEFA + LPS + NLRP3 inhibitor groups. Levels and activation of pyroptosis-related markers (NLRP3, ASC, Caspase-1, GSDMD, IL-18 and IL-1β) were measured. <b>Results</b>: NEFAs alone upregulated these markers in a dose-dependent manner. Compared to NEFAs alone, NEFA + LPS co-treatment significantly enhanced levels of the markers, increased IL-1β secretion, and promoted NLRP3/Caspase-1 co-localization and Caspase-1activity. Notably, these effects of NEFA + LPS were attenuated by the NLRP3 or Caspase-1 inhibitors. Similar results were obtained when repeating the experiments in carcinoma HepG2 cells. Also, a random liver section from the subclinical ketotic cows displayed a higher fluorescence intensity of NLRP3 and Caspase-1 and stronger co-localization than that from a healthy cow. <b>Conclusions</b>: NEFAs and LPS synergistically contribute to pyroptosis in bovine hepatocytes by enhancing NLRP3 inflammasome assembly and subsequent Caspase-1 activation, providing a potential target for mitigating hepatic injury.