Absorption, transport, blood-brain barrier penetration, and neuroprotection of walnut peptides LR and LPI

The prerequisite for neuroprotective peptides to exert physiological effect is that they can across intestinal epithelial barrier and blood-brain barrier (BBB). The study was aimed to investigate the absorption, transportation and BBB permeability of walnut neuroprotective peptides LR and LPI using Caco-2 cell monolayer and in vivo imaging, and further to evaluate their underlying mechanisms through transcriptome sequencing analysis of zebrafish brain. Results showed that LR and LPI improved learning and memory impairment in scopolamine-induced zebrafish. Both peptides could be intactly transported in Caco-2 cells but via different mechanisms. LR was transported via both PepT1-mediated active route and tight junction-regulated passive paracellular route, while LPI was via PepT1 route only, with apparent permeability coefficient (30.18 ± 1.94) × 10⁻⁷ cm/s and (51.91 ± 3.49) × 10⁻⁷ cm/s, respectively. In vivo imaging of nude mice after FITC-labeling peptides administration further consolidated their ability of absorption, metabolic stability, and BBB penetration. Interestingly, LR exhibited better brain influx than that of LPI in nude mice. Additionally, transcriptome sequencing analysis demonstrated that LR and LPI improved learning and memory capacity by intervening cholinergic system, synaptic development and plasticity, neurotrophins, and oxidative stress, which were subsequent verified by biochemical measurement of zebrafish brain.