Walnut-Derived Peptide Enhances Mitophagy via JNK-Mediated PINK1 Activation to Reduce Oxidative Stress in HT-22 Cells

Mitophagy has a neuroprotective effect on reactive oxygen species (ROS)-induced neurodegenerative diseases. The walnut-derived polypeptide (TW-7) has antioxidant activity and protects nerves by promoting autophagy. However, its action mechanism against oxidative stress through mitophagy remains obscure. Therefore, we aimed to assess the effects of TW-7 on HT-22 cells under oxidative stress. Mitochondrial ultrastructure and cristae number were observed by transmission electron microscopy. The results showed that TW-7 (100 μM) restored the fluorescence intensity of the mitochondrial membrane potential to 0.99 ± 0.04 (P < 0.05), decreased H₂O₂-induced opening of mitochondrial permeability transition pores, and inhibited mitochondrial bioenergetic deficits. Moreover, it significantly increased activities of antioxidant enzymes to 186.88 ± 5.40 U/mgprot, 40.08 ± 0.87 mU/mgprot, and 23.57 ± 0.77 U/mgprot (P < 0.05), based on superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) assay results, respectively. Consistently, it decreased cellular and mitochondrial ROS levels by 51.71 ± 0.81 and 49.75 ± 0.69% (P < 0.05). TW-7 also downregulated C-Jun N-terminal kinase (JNK) phosphorylation and activated PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy in H₂O₂-induced HT-22 cells treated with JNK activator (anisomycin) and inhibitor (SP600125). Furthermore, TW-7 inhibited the mitochondrial apoptosis pathway by downregulation of the cytoplasmic cytochrome C, caspase-9, and cleaved-caspase-3 expression. Additionally, BDNF and SNAP-25 levels significantly increased to protect the synaptic function. Collectively, TW-7 improved oxidative stress-mediated nerve cell injury via JNK-regulated PINK1-mediated mitophagy.