Caveolin Regulates the Transport Mechanism of the Walnut-Derived Peptide EVSGPGYSPN to Penetrate the Blood-Brain Barrier.

Bioactive peptides, derived from short protein fragments, are recognized for their neuroprotective properties and potential therapeutic applications in treating central nervous system (CNS) diseases. However, a significant challenge for these peptides is their ability to penetrate the blood-brain barrier (BBB). EVSGPGYSPN (EV-10) peptide, a walnut-derived peptide, has demonstrated promising neuroprotective effects in vivo. This study aimed to investigate the transportability of EV-10 across the BBB, explore its capacity to penetrate this barrier, and elucidate the regulatory mechanisms underlying peptide-induced cellular internalization and transport pathways within the BBB. The results indicated that at a concentration of 100 μM and osmotic time of 4 h, the apparent permeability coefficient of EV-10 was Papp = 8.52166 ± 0.58 × 10 cm/s. The penetration efficiency of EV-10 was influenced by time, concentration, and temperature. Utilizing Western blot analysis, immunofluorescence, and flow cytometry, in conjunction with the caveolin (Cav)-specific inhibitor M-β-CD, we confirmed that EV-10 undergoes transcellular transport through a Cav-dependent endocytosis pathway. Notably, the tight junction proteins ZO-1, occludin, and claudin-5 were not disrupted by EV-10. Throughout its transport, EV-10 was localized within the mitochondria, Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, and cell membranes. Moreover, Cav-1 overexpression facilitated the release of EV-10 from lysosomes. Evidence of EV-10 accumulation was observed in mouse brains using brain slice scans. This study is the first to demonstrate that Cav-1 can facilitate the targeted delivery of walnut-derived peptide to the brain, laying a foundation for the development of functional foods aimed at CNS disease intervention.