The Carnosine–HNE Michael Adduct as a Redox-Active Species Associated with Nrf2-Dependent Antioxidant and Anti-Inflammatory Responses

Carnosine (CAR), an endogenous histidine-containing dipeptide, exhibits antioxidant and anti-inflammatory activity in various experimental models; however, its molecular mechanism of action remains poorly understood. Here, we demonstrate that the Michael adduct between CAR and 4-hydroxy-2-nonenal (HNE), which has been detected in previous studies in both in vitro and in vivo settings, mediates its bioactivity, particularly its antioxidant and anti-inflammatory responses, through Nrf2 activation. The CAR–HNE adduct was synthesized and its physicochemical, metabolic, and biological properties were evaluated. CAR–HNE exhibited high stability in biological matrices and retained the ability to transfer HNE to thiol nucleophiles at a slow rate under physiologically relevant conditions, consistent with electrophile-mediated Nrf2 activation. This kinetic behavior limits the cytotoxicity typically associated with free HNE while preserving the redox signaling capacity. CAR–HNE induced dose-dependent Nrf2 activation and NF-κB inhibition in cell-based assays without the hormetic toxicity observed for free HNE. Mechanistically, CAR–HNE may act as a redox-tunable electrophilic reservoir, restoring nucleophilic tone and modulating redox-sensitive transcription factors. In vivo, CAR–HNE attenuated DSS-induced colitis more effectively than equimolar doses of either carnosine or HNE alone. Proteomic analyses revealed modulation of canonical Nrf2-dependent antioxidant pathways. Our findings suggest a conceptual shift in carnosine biology: rather than acting as a classical antioxidant or carbonyl quencher, carnosine functions as a precursor of redox-active electrophilic adducts that transduce anti-inflammatory and antioxidant responses via controlled RCS signaling.