Berberine inhibits muramyl dipeptide-induced oxidative stress and pyroptosis in canine corneal epithelial cells via the NOD2 signaling pathway.

Bacterial keratitis poses a significant threat to ocular health in companion animals, particularly dogs, often leading to corneal ulceration and visual impairment. Muramyl dipeptide (MDP), a conserved bacterial peptidoglycan motif, activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2), yet its contribution to corneal pathogenesis remains incompletely defined. This study demonstrated that MDP induced the NOD2-dependent activation of the RIPK2/NF-κB pathway, triggering pro-inflammatory cytokine production and suppressing the Nrf2-mediated antioxidant response, thereby exacerbating oxidative stress in canine corneal epithelial cells. Furthermore, MDP promoted NLRP3 inflammasome assembly, caspase-1 activation, and gasdermin D-mediated pyroptosis. Inhibition of NOD2 signaling attenuated these effects, highlighting its central role in coordinating inflammatory and oxidative damage. Notably, the natural compound berberine (BBR) effectively suppressed NOD2 activation, restored redox homeostasis, and inhibited pyroptosis in both cellular and canine models of MDP-induced keratitis. These results revealed a previously unrecognized mechanism by which MDP exacerbated corneal damage through NOD2-driven integration of oxidative stress and pyroptosis. We also identified BBR as a multi-target therapeutic agent capable of disrupting this pathogenic cascade. Our findings provided mechanistic insights into the treatment of bacterial keratitis through modulation of innate immune signaling pathways.