Neuroprotection by Walnut-Derived Peptides through Autophagy Promotion via Akt/mTOR Signaling Pathway against Oxidative Stress in PC12 Cells

Natural-derived peptides are effective substances in attenuating oxidative stress. However, their specific mechanisms have not been fully elucidated, especially in peptide-mediated autophagy. In the present study, TWLPLPR, YVLLPSPK, and KVPPLLY, novel peptides from Juglans mandshurica Maxim, prevented reactive oxygen species (ROS) production, elevated glutathione peroxidase (GSH-Px) activity and adenosine 5'-triphosphate (ATP) levels, and ameliorated apoptosis in Aβ_25-35 (at a concentration of 50 μM for 24 h)-induced PC12 cells (P < 0.01). Both western blot and immunofluorescence analysis illustrated that the peptides regulated Akt/mTOR signaling through p-Akt (Ser473) and p-mTOR (S2481) and promoted autophagy by increasing the levels of LC3-II/LC3-I and Beclin-1 while lowering p62 expression (P < 0.01). The autophagy inhibitor (3-methyladenine, 3-MA) and inducer (rapamycin, RAPA) were combined used to confirm the contribution of peptide-regulated autophagy in antioxidative effects. Moreover, the peptides increased the levels of LAMP1, LAMP2, and Cathepsin D (P < 0.05) and promoted the fusion with lysosomes to form autolysosomes, accelerating ROS removal. These data suggested that walnut-derived peptides regulated oxidative stress by promoting autophagy in the Aβ_25-35-induced PC12 cells.