Honokiol ameliorates pyroptosis in bovine mammary epithelial cells via SIRT3-mediated inhibition of the NLRP3 inflammasome.

Bovine mastitis causes significant economic losses in the dairy industry, with excessive inflammation and pyroptosis in its pathogenesis. Honokiol (HKL), a natural compound that may regulate mitochondrial deacetylase Sirtuin 3 (SIRT3)-related signaling, possesses anti-inflammatory properties. This study aimed to investigate whether HKL exerts therapeutic efficacy against lipopolysaccharide (LPS)-induced pyroptosis and to explore its regulatory mechanism. In vitro, Western blot and ELISA were employed to assess pyroptosis markers and cytokine release in LPS-stimulated bovine mammary epithelial cells (MAC-T) and human breast cancer cells (MCF-7) treated with HKL. Furthermore, SIRT3 knockdown was established via siRNA transfection, followed by the assessment of NLRP3 inflammasome assembly via colocalization. In vivo, HKL efficacy in a mouse mastitis model was confirmed by histopathology and Western blot. HKL significantly inhibited LPS-induced pyroptosis in MAC-T and MCF-7 cells, as evidenced by downregulated expression of NLRP3 inflammasome components (NLRP3, GSDMD, Caspase-1, Caspase-1 p20 and ASC) and reduced secretion of pro-inflammatory cytokines IL-1β and IL-18. Crucially, HKL enhanced SIRT3 expression, whereas SIRT3 silencing largely abrogated the anti-pyroptotic effects of HKL and reactivated NLRP3 inflammasome assembly. Consistent with in vitro findings, HKL administration remarkably alleviated mammary tissue damage and suppressed pyroptosis in the mouse model. In summary, HKL ameliorates pyroptosis via SIRT3-mediated inhibition of the NLRP3 inflammasome. This study supports HKL as a potential precision therapy for bovine mastitis and highlights SIRT3 as a pivotal regulatory target for controlling mammary inflammation.