Compound 3a attenuates myocardial ischemia-reperfusion injury by inhibiting IKK β -dependent oxidative stress

Oxidative stress represents a pivotal pathological driver in ischemia/reperfusion (I/R) injury. Compound 3a, derived from nordihydroguaiaretic acid and alpha-β-unsaturated ketone scaffolds, exhibits strong antioxidant properties. Systematic evaluation across in vitro hypoxia/reoxygenation (H/R) models and in vivo I/R injury paradigms revealed that 3a robustly enhances cellular antioxidant defenses. Mechanistically, 3a administration (5 μM) significantly attenuated H/R-induced upregulation of phosphorylated IκB kinase (p-IKKβ) and cylindromatosis (CYLD) while elevating nuclear factor erythroid 2-related factor 2 (NRF2) levels by 1.6-fold (p < 0.01), indicating activation of endogenous antioxidant pathways. Immunoprecipitation identified Kelch-like ECH-associated protein 1 (Keap1) as a direct substrate of CYLD, revealing its role in oxidative defense regulation. In addition, 3a reduces damage to the body and the damage that oxidative stress causes in the heart. Quantitative analysis revealed a significant 38 ± 6 % elevation in myocardial superoxide dismutase (SOD) activity (p < 0.05), indicative of enhanced endogenous antioxidant capacity, concomitant with a 34 ± 3 % reduction in serum creatine kinase-myocardial band (CK-MB) levels (p < 0.01). Our findings suggest that 3a pretreatment activates KEAP1-NRF2 signaling, reducing oxidative stress and potentially preventing cardiac ischemia/reperfusion injury in vitro and in vivo. Thus, 3a presents a promising therapeutic strategy for developing drugs to mitigate cardiac ischemia-reperfusion injury.