Improving Study Of Walnut Peptide On Memory And Cognitive Deficits, And Its Absorption, Metabolism And Mechanism

Ageing population and fast-paced lifestyle led to a steady increase in the prevalence of neurodegenerative diseases associated with memory and cognitive impairment,which are gradually becoming prevalent in the young group.In recent years,bioactive peptides attract much attention due to their wide range of sources,simple preparation process,easy absorption,potent biological activity and no side-effects.Widespread research has proven that bioactive peptides have promising potential in learning and memory improvement.The purpose of this thesis was to determine the ameliorative effect of walnut protein hydrolysate（WPH）against memory deficits induced by scopolamine and further to validate the memory-improving efficacy of target peptides identified and screened by integrated approaches of peptidomics and bioinformatics.Subsequently,the in vitro/in vivo absorption,metabolism,blood-brain barrier penetration,and underlying memory-improving mechanism of target walnut peptides were investigated.Finally,based on the crucial role of arginine residues in neuroprotective peptides,and the positive effects of walnut Arg-containing peptide LR on absorption and transport,blood-brain barrier permeability,and brain transcriptomic regulations observed in this study.Arg-containing peptides enriched WPHs were prepared by in silico hydrolysis and controlled enzymatic release,and then validated their efficacy.This thesis can provide theoretical basis and technical guidance for walnut protein deep processing and high value-added utilization,as well as memory improvement peptides development.The main contents and results are shown as follows:（1）WPH was prepared through bio-enzymatic hydrolysis of walnut protein,and its ameliorative effect on scopolamine-induced memory and cognitive impairment was investigated by evaluating behavioral performance in Morris water maze trails and biochemical indicators in mice brain.Results showed that WPH dose-dependently enhanced the behavioral performance of scopolamine-elicited memory dysfunction mice in water maze test,and also ameliorated scopolamine-induced neuronal pathological changes in the brain.Further biochemical analysis exhibited that WPH could enhance memory and cognitive function by modulating cholinergic system,oxidative stress and neurotrophic pathways.（2）The purpose of this chapter was to identify the peptide profile of WPH and further to screen target peptides by combining peptidomic and bioinformatic approaches.Subsequently,the efficacy of target walnut peptides was verified in scopolamine-induced memory impairment zebrafish.Results showed that a total of 180 peptides were identified from WPH,and then the promising neuroprotective peptides were filtered based on the comprehensive consideration of protein database matching,relative peak areas,Peptide Ranker scores,and molecular docking affinity with the target proteins.Among them,target peptides LR and LPI significantly relieved the memory deficits of zebrafish,providing a substrate basis for further studying of walnut peptides on absorption,metabolism and mechanism in vitro and in vivo.（3）The uptake mechanism of LR and LPI was investigated across intestinal epithelium using human intestinal Caco-2 cell monolayers,and then their absorption,metabolism and capability to cross blood-brain barrier were systematically evaluated using in vivo imaging techniques.Results showed that both LR and LPI were transported intactly across intestinal epithelium with apparent permeability coefficients of（30.18±1.94）×10^-7 cm/s and（51.91±3.49）×10^-7 cm/s,respectively,speculating that their transport route might be closely related to the Pep T1-mediated active transport,energy-dependent transcytosis and tight junction-regulated paracellular diffusion.In vivo imaging study exhibited that LR and LPI had high absorption capacity and metabolic stability in nude mice,and further imaging analysis of ex vivo mice brain also confirmed that LR and LPI could cross blood-brain barrier.（4）Transcriptomic analysis was used to evaluate the differences of transcription profile of zebrafish brain to explore the effect of LR and LPI on scopolamine-induced memory and cognitive impairment,and further to analyze and validate the signal pathways by conjunction with brain biochemical measurements for revealing their underlying mechanisms.Transcriptomic studies showed that LR and LPI mainly regulated cholinergic system,neurotransmitters and synapses,oxidative stress and neurotrophic pathways,and the genes related to above pathways were located at the center of the target differentially expressed gene regulatory network in protein-protein interaction analysis.Subsequently,results of brain biochemical measurements further supported the reliability of the transcriptomic analysis.（5）On the basis of the clear ameliorative effect of walnut-derived Arg-containing peptides on scopolamine-induced memory and cognitive dysfunction in zebrafish.Integrated approaches of in silico simulated hydrolysis and controlled enzymatic release were applied to prepare WPHs enriched with Arg-containing peptides,and then their efficacy was validated as well.Arg-containing peptides designed from walnut storage protein sequences with relative high abundance,Peptide Ranker scores,and CPPpred scores significantly improved scopolamine-induced memory impairment.Notably,the presence of arginine in the form of peptide rather than free arginine contributed greater neuroprotection.Meanwhile,the positive charge and cell-penetrating properties also affected the neuroprotection of Arg-containing peptides.Subsequently,WPHs enriched with Arg-containing peptides significantly ameliorated scopolamine-elicited memory deficits in zebrafish,indicating that the combination of in silico hydrolysis and controlled enzymatic release might be an effective approach to obtain Arg-containing neuroprotective peptides.