The crosstalk between sequestosome 1 and heme oxygenase-1 in regulating autophagy/oxidative stress in bovine mammary epithelial cells

Bovine mastitis severely compromises dairy cattle health, primarily through oxidative stress and dysregulated autophagy in bovine mammary epithelial cells (BMECs), ultimately impairing lactation performance. While heme oxygenase-1 (HO-1) is well-established as a critical regulator of oxidative stress and autophagy, its interplay with the selective autophagy receptor sequestosome 1 (SQSTM1/p62), as a key mediator linking autophagy to antioxidant responses, remains unclear in the context of mastitis. We hypothesized that p62 modulates HO-1 activity to coordinate cellular defense mechanisms in BMECs. To test this, we established p62 overexpression/knockdown cell lines, treating them with lipopolysaccharide (LPS), exogenous HO-1, nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, or autophagy inhibitor. Our results demonstrated that p62 overexpression suppressed autophagy while concurrently activating the Nrf2/HO-1 pathway by binding to kelch-like ECH-associated protein 1 (Keap1). The p62-HO-1 axis and exogenous HO-1 synergistically enhanced antioxidant enzyme expression, attenuated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and mitigated LPS-induced pro-inflammatory cytokine production. Mechanistically, the p62-HO-1 axis restored mitochondrial membrane potential, elevated adenosine triphosphate (ATP) synthesis, reduced mitochondrial superoxide accumulation, and corrected mitochondrial dynamics imbalances. Crucially, these protective effects were found to be autophagy-independent but Nrf2-dependent. This study elucidates a novel mechanism by which the p62-HO-1 axis safeguards BMECs by integrating antioxidant defenses with mitochondrial homeostasis, offering new insights into the pathogenesis of bacterial mastitis and identifying potential therapeutic targets.