Study On Memory-improving Effects Of Round Scad Peptide On Sleep Deprivation-induced Memory Impairment

In recent years,lack of sleep or descended sleep quality has become a common social phenomenon affecting human health.Lack of sleep is often accompanied by memory and cognitive decline,and may even lead to neurodegenerative diseases.Studies have shown that food-based intervention strategies can help prevent and delay memory impairment.Bioactive peptides are considered to be natural bioactive ingredients for the prevention or control of chronic diseases and promoting human health due to their wide range of sources,easy digestion and absorption,multiple physiological functions as well as safety and effectiveness.In this study,neuroprotective protein hydrolysate derived from round scad was prepared by controlled enzymatic hydrolysis technology.The memory-improving effects of neuroprotective hydrolysate in vivo were evaluated by animal models.Then,neuroprotective peptides were separated and identified from the round scad hydrolysate(RSH),followed by the evaluation of their neuroprotection on glutamate-induced excitotoxicity in cells,and we further evaluated the memory-improving effects of peptides in sleep-deprived mice and explored the underlying mechanisms.The effects of the gastrointestinal digestive process on the structure and activity of neuroprotective peptides were further evaluated,and the absorption and structure-activity relationship of neuroprotective peptides were also analyzed.This study provides a theoretical basis and technical guidance for the production,development,and application of memoryimproving peptides.The main research contents and results of this study are as follows:(1)Pancreatin and protamex were used to prepare the round scad hydrolysate with different degrees of hydrolysis(6%,12%,18%,and 24%).The results showed that RSH was dominated by small molecular peptides,and this part gradually increased with the increase of the degree of hydrolysis.RSH was rich in essential amino acids,hydrophobic and aromatic amino acids.Furthermore,with the increase of the degree of hydrolysis,antioxidant and neuroprotective effects of RSH firstly increased and then decreased.RSH with a degree of hydrolysis of 18%(RSH-18)had the highest antioxidant and neuroprotective effects.Furthermore,RSH-18 ameliorated the excitotoxicity induced by glutamate,increased cell survival,and inhibited cell membrane damage.In addition,oral administration of RSH-18 could significantly improve sleep deprivation-induced spatial memory impairment in rats.(2)Taking cell viability of glutamate-damaged neuronal cells as an indicator,ultrafiltration technology combined with sephadex G-15 gel filtration were used to separate the RSH-18,and then 23 peptides were identified by UPLC-QTOF-MS/MS,which were characterized by high content of aromatic amino acids and hydrophobic amino acids.These identified peptides were then synthesized and their neuroprotection were evaluated in glutamate-induced cellular model.Among them,octapeptide WCPFSRSF exerted the highest neuroprotection.Further studies showed that WCPFSRSF significantly increased the activities of antioxidant enzymes,increased the levels of endogenous antioxidants,and decreased the level of MDA.In addition,WCPFSRSF up-regulated the protein expression levels of Nrf2 and its target genes NQO1 and HO-1,promoting the nuclear translocation of Nrf2.Moreover,WCPFSRSF inhibited the expression of NFκB and the degradation of IκB-α,and octapeptide was involved in the regulation of the interaction between Nrf2 and NFκB.(3)Effects of octapeptide WCPFSRSF on memory impairment induced by sleep deprivation in mice were studied.The results of behavior test showed that oral administration of WCPFSRSF ameliorated sleep deprivation-induced spatial memory impairment in mice.Furthermore,WCPFSRSF ameliorated sleep deprivation-induced pathological changes in the hippocampus.WCPFSRSF also ameliorated oxidative stress injury,increased the antioxidant defense system.Transcriptomic studies showed that WCPFSRSF mainly regulated inflammation-related pathways,neurotransmitter systems,and neurotrophic-related pathways.Neuroinflammation-related genes were located at the center of the protein-protein interaction(PPI)network.Further analysis showed that WCPFSRSF inhibited the activation of microglia and astrocytes in the hippocampus.Additionally,WCPFSRSF inhibited the activation of IL-6/STAT/JAK pathway and inflammasome in hippocampus.(4)Effects of in vitro simulated gastrointestinal digestion on the octapeptide WCPFSRSF were explored.The results showed that INFOGEST in vitro simulated gastrointestinal digestion slightly reduced the neuroprotective activity of WCPFSRSF.The changes of octapeptide during gastrointestinal digestion were further analyzed by UPLC-QTOF-MS/MS,and the results showed that the octapeptide was less than 1% of the initial amount at the end of gastrointestinal digestion.Subsequently,eleven and nine peptides were identified from final gastric digestion production of WCPFSRSF(W-G)and final gastrointestinal digestion production of WCPFSRSF(W-GI),respectively,with m/z ranging from 262.1510 to 1029.4610.To further analyze the differences of the degraded peptides,bioinformatics combined with molecular simulation analysis were used to explore the characteristics of the peptides identified in W-GI.Results showed that these peptides were mainly divided into three groups: the first group were WCP,WCPF,WCPFS,PF and PFS;the second group were SR and SRS;and the third group was WCPFSRS.In vitro cell experiments showed that two representative peptides,SR and WCPF,could significantly improve the cell survival in glutamate-damaged SH-SY5 Y cells,and the neuroprotective effect of WCPF was stronger than that of SR.(5)Based on the results of animal experiments and peptides identified from the W-GI,the absorption and anti-neuroinflammatory of neuroprotective peptides,as well as their structureactivity relationship were further evaluated.The results showed that SR,WCP and WCPF could be absorbed and transported by Caco-2 cell monolayer in an intact form of peptides.However,WCPFS was mainly absorbed by Caco-2 cells in the form of its degraded peptide WCPF.We further explored the regulatory role of above three peptides on neuroinflammation,it was found that all three peptides could inhibit the release of inflammatory mediators and cellular inflammatory factors,and regulate the TLR4/NFκB and JAK2/STAT3 pathways.Their ability to inhibit neuroinflammation was ranked as WCPF > WCP > SR.The analysis of the structureactivity relationship showed that the anti-neuroinflammatory role of SR may be associated with Arg at C-terminal,while the anti-neuroinflammatory role of WCP and WCPF may be associated with Trp at N-terminal.