Methyl eugenol attenuates age-associated oxidative fragility by coupling Ca²⁺-calpain inhibition with Band 3 stabilization in human erythrocytes

BACKGROUND: Human erythrocytes serve as an ideal model for cellular aging, a process where longevity relies on membrane scaffold integrity. The oxidative deterioration of Band 3, a major integral membrane protein, is a central driver of this senescence. This study investigated whether methyl eugenol (ME) stabilizes Band 3 against age-associated oxidative fragility. METHODS: Erythrocytes were challenged with H₂O₂ to simulate age-associated oxidative injury. Damage was evaluated via hemolysis assays, SEM, and flow cytometry. Sulfate (SO₄ ²⁻) uptake kinetics and Western blotting were employed to assess Band 3 anion exchange function and structural stability. In silico docking simulated interactions between ME metabolites and the Band 3 structure. Physiological relevance was validated in a human cohort (n = 81; 20-90 years) via regression and stratified analyses of glutathione (GSH) and malondialdehyde (MDA) levels. RESULTS: ME exhibited an optimal protective concentration at 2 µM, effectively preserving biconcave morphology and attenuating hemolysis. Treatment significantly mitigated intracellular oxidative stress and rescued cell viability. Mechanistically, ME suppressed the pathological increase in intracellular Ca²⁺ concentration and inhibited calpain activity. Functionally, ME significantly restored sulfate transport rates. Western blotting confirmed that ME specifically preserved the full-length (100 kDa) and cytoplasmic (43 kDa) domains of Band 3, whereas the 55 kDa transmembrane domain remained largely unaffected. Docking simulations predicted a specific interaction with residue ARG292 within the cytoplasmic domain, suggesting a structural basis for this stabilization. In the donor cohort, ME extended the projected GSH half-life (from 47.14 to 64.14 years) and reduced maximal lipid peroxidation by ~40%. CONCLUSION: ME mitigates oxidative eryptosis by coupling Ca²⁺-calpain inhibition with site-specific Band 3 stabilization, offering a rationale for using ME to standardize erythrocyte quality and reduce age-associated fragility.