Combined Antioxidant Effects of Kaempferol and N-Acetylcysteine on H2O2-Induced Oxidative Stress in SH-SY5Y Cells

This study investigated the combined antioxidant effects of kaempferol (KAE) and N-acetylcysteine (NAC) against hydrogen peroxide (H2O2)-induced oxidative stress in SH-SY5Y cells. Combination effects were evaluated using combination index (CI) analysis and SynergyFinder. Cell viability, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence, malondialdehyde (MDA), antioxidant enzyme activities, glutathione status, and protein expression of sirtuin 3 (SIRT3), forkhead box O3 (FOXO3a), and superoxide dismutase 2 (SOD2) were assessed. Molecular docking was performed to predict potential interactions of KAE and NAC with SIRT3. The strongest CI-defined synergy occurred at 16 μM KAE and 2 mM NAC (CI = 0.10), whereas SynergyFinder results varied across reference models. The combination (KNAC) enhanced cell viability, reduced CM-H2DCFDA fluorescence and MDA levels, improved antioxidant enzyme activities and glutathione redox balance, and increased SIRT3, FOXO3a, and SOD2 protein levels compared with individual treatments. Molecular docking predicted a more favorable binding score for KAE than NAC against SIRT3. These findings suggest that KAE and NAC may produce complementary antioxidant and cytoprotective effects through potentially distinct mechanisms, with KAE potentially contributing to changes in proteins associated with mitochondrial antioxidant defense and NAC supporting glutathione-dependent cellular redox homeostasis. However, the causal involvement of the SIRT3/FOXO3a/SOD2 pathway and direct interactions with SIRT3 remain to be experimentally confirmed. These preliminary findings support further investigation of combining food-derived KAE with NAC as a glutathione precursor to enhance cellular antioxidant defenses.