Suo_2026_Mar.Drugs_24_

Although the Pacific oyster (Crassostrea gigas) is a premium marine protein source, its neuroprotective peptidome remains largely uncharacterized. This study established an integrated in silico and in vitro pipeline to discover acetylcholinesterase (AChE)-targeting peptides with cellular AChE-regulating and neuroprotective peptides from simulated gastrointestinal digests of oyster. Peptidomic profiling identified 18,292 sequences, which were filtered down to seven candidates predicted to have favorable blood-brain barrier (BBB) permeability and to be non-toxic and non-allergenic (VPYPR, VPVHF, HHTF, PVHF, GPKPW, HWF, and KYW) via multi-step virtual screening. In cellular assays, simulated H(2)O(2) injury (500 microM) reduced PC12 cell viability to 47.53 +/- 4.53%. Compared with the model group, pretreatment with the three most potent candidates-HHTF, VPYPR, and VPVHF (200 microM)-significantly rescued injured cells, restoring cell viability to 88.31 +/- 7.83%, 85.12 +/- 3.35%, and 82.00 +/- 3.47%, respectively (p < 0.05). These peptides effectively fortified cellular antioxidant defenses by increasing glutathione (GSH) levels to 24.24, 30.11, and 26.83 nmol/mg protein (from 20.22 nmol/mg protein in the model group) and superoxide dismutase (SOD) activity to 151.41, 153.97, and 151.96 U/mg protein (from 119.33 U/mg protein), while suppressing malondialdehyde (MDA) accumulation to 0.088, 0.064, and 0.086 nmol/mg protein (from 0.193 nmol/mg protein). Crucially, the peptides alleviated cholinergic dysfunction by normalizing the H(2)O(2)-induced elevation of intracellular AChE activity (11.39 nmol/min/mg protein) down to 7.02, 6.22, and 7.14 nmol/min/mg protein, respectively. Specifically, VPYPR (200 microM) restored AChE activity to a level (6.22 nmol/min/mg protein) that was not significantly different from that in the normal control group (p > 0.05). Molecular dynamics (MD) simulations (100 ns) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations identified VPYPR as the leading candidate with a remarkably low binding free energy of -49.74 +/- 3.58 kcal/mol. This study demonstrates that oyster gastrointestinal digests are valuable reservoirs of multi-target neuroprotective ingredients and provides an efficient strategy for marine bioactive peptide discovery.