Yak Casein Peptides Exhibit Antioxidant Activity via Regulating Keap1-Nrf2/ARE Pathway in H ₂ O ₂ -Induced HEK-293 Cells

In this study, the antioxidant mechanisms of yak casein-derived antioxidant peptides (YCAPs: AFK, IEQI, FPFF, LPVPQ, RELEEL) were systematically investigated using an H₂O₂-induced HEK-293 cell model. Treatment with YCAPs at a concentration of 200 μg/mL significantly improved cell viability and enhanced cellular antioxidant capacity, as indicated by increased activities of CAT and SOD to 0.60 ± 0.094 and 1.17 ± 0.016 U/10⁴ cells, respectively. Concurrently, MDA and GSSG levels were reduced to 0.062 ± 0.0004 nmol/10⁴ cells and 0.74 ± 0.26 μg/10⁶ cells, respectively (<i>P</i> < 0.05). Furthermore, YCAPs upregulated the expression of Nrf2, HO-1, and NQO1, while downregulating Keap1 expression under oxidative stress conditions <i>in vitro</i> (<i>P</i> < 0.05). Molecular docking analysis revealed strong binding affinities between YCAPs and Keap1, with binding energies ranging from -7.8 to -10.1 kcal/mol, suggesting that YCAPs interfere with the protein-protein interaction between Keap1 and Nrf2. These findings indicate that YCAPs exert their antioxidant effects primarily through activation of the Keap1-Nrf2/ARE signaling pathway.