The Carnosine-HNE Michael Adduct as a Redox-Active Species Associated with Nrf2-Dependent Antioxidant and...

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.