Donepezil Reduces H₂O₂-Inflicted Oxidative Stress and Necroptosis in Cardiomyocytes

Necroptosis, as a form of regulated cell necrosis, could participate in myocardial oxidative damage. We investigated whether donepezil attenuates H₂O₂-induced oxidative stress injury and necroptosis in rat cardiomyocytes.

H9c2 cells were incubated with H₂O₂ (final concentration of 1 mM) and then intervened with donepezil at doses of 2.5 and 10 μM. Subsequently, the necroptosis inhibitor necrostatin-1 (Nec-1) was introduced to treat H9c2 cells. For cell function experiments, cell proliferation; the contents of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA); the protein and mRNA levels of the necroptosis-related proteins receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase-like (MLKL); and calcium ion fluorescence intensity were detected using Cell Counting Kit-8, enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative reverse transcription polymerase chain reaction, and flow cytometry, respectively.

Cell viability was conspicuously decreased; CK and LDH contents, RIP3 and MLKL expression levels, and MDA production were preeminently elevated; and the production of SOD, CAT, and GSH was prominently reduced under H₂O₂ stimulation, which were dose-dependently countered by donepezil intervention. Nec-1 decreased the cell necroptosis, oxidative stress, and calcium overload caused by H₂O₂. However, on the premise of donepezil intervention, the addition of Nec-1 failed to further improve the situation, suggesting that donepezil exerts cardioprotective effects partly by inhibiting RIP3 and MLKL levels.

Donepezil reduced H₂O₂-inflicted oxidative stress and necroptosis in cardiomyocytes by suppressing RIP3 and MLKL levels and calcium ion overload.