Study Of The Learning And Memory-improving Mechanisms Of Walnut Protein-derived Bioactive Peptide

Food-derived bioactive peptides are functional factors possessing abundant biological activities with great development prospects.Walnut has long been widely used as a functional food for brain health;however the underlying mechanisms remain unknown.Although unsaturated fatty acids and vitamins are regarded as major bioactive components of Walnut involved in brain health,proteins and its-constituent bioactive peptides are also considered as major components.In comparison with walnut protein,walnut-derived peptides have attracted much attention due to its low molecular weight and higher absorption rate.The aim of this study was to screen bioactive peptides possessing cognitive function improvement ability from walnut protein hydrolysates(WPH).Then we investigated their anti-beta-amyloid(A?)protein aggregation activity in a cell model,constructed with A?42-mCherry(E22G)plasmid leading to A? aggregation.Cognitive impairment-improving activity was also investigated in vivo.Finally,we explored the possible mechanisms and administration routes for the bioactive peptide.In the present work,we first investigated the effects of WPH on learning and memory impairments in D-gal induced mice.The behavioral results showed that WPH could ameliorate learning and memory impairments to spatial and harmful stimuli in D-gal induced mice.We found that WPH significantly improved the levels of antioxidase(SOD and GSH-Px)activity,inhibited NOS activity,and reduced oxidative products(MDA and NO)in serum and brain tissues.Furthermore,WPH could remarkably inhibit the aggregation of A? protein in cell model.It was found that the molecular weight distribution of WPH peptides as detected by HPLC were mainly below 1 500 Da.Ten of such peptides were successfully identified from WPH using LC-MS/MS,and their sequences were as follows: ASACM(AM5),PPKNW(PW5),ASSAPE(AE6),GGPATTC(GC7),HCPF(HF4),HSGPHA(HA6),NGGSH(NH5),WSREEQE(WE7),MTDAN(MN5)and WPPKN(WN5).Four peptides,including WN5,PW5,WE7 and HF4,were then selected to explore their anti-A? aggregation activity in vitro.Peptide PW5 showed the best anti-A? aggregation activity among the rest.To further investigate the biological activity of peptide PW5 in vivo,APP/PS1 transgenic mouse model with cognitive impairments was adapted.The results of behavioral tests showed that PW5 could improve learning and memory ability to spatial and harmful stimuli in the Morris water maze and Shuttle box test,respectively.Immunohistochemistry staining demonstrated that PW5 could significantly reduce A? plaques in the brain(15.5 ± 1.08,p <0.05)compared to vehicle treated mice(25.14 ± 3.89).In addition,we also found that PW5 could alter the diversity and compositions of gut microbiome imbalance in APP/PS1 mice,and also increased remarkably serum norepinephrine(NE)level and decreased the levels of Acetylcholine(Ach)and butyric acid(p < 0.05).Pearson correlation analysis showed that the alterations in serum neurotransmitters and short-chain fatty acids levels were correlated with changes in relative abundance of several genera including Lactobacillus,g_norank_f_Erysipelotrichaceae and Flexispira.The role of the gut microbiota in the development of AD upon PW5 administration was explored in APP/PS1 mice.It has been found that short-term antibiotic cocktail(Ampicillin,Metronidazole,Vancomycin,Neomycin,Gentamicin and Erythromycin)treatment could effectively deplete most of the gut bacteria in APP/PS1 mice to obtain a near germ-free status,without impacting on A? deposition.Mice treated with antibiotic cocktail were then transplanted with fecal microbiota from aged(16 months old)APP/PS1 transgenic mice.The results showed that FMT could reconstitute the gut microbiota community and led to a significant increase in A? plaques(p < 0.05).By comparing the effects of oral administration and intraperitoneal injection of PW5 on learning and memory impairment in APP/PS1 mice,it was found that oral administration of peptide PW5 could effectively ameliorate cognitive impairments and reduce A? deposits in APP/PS1 mice better than intraperitoneal injection.Moreover,in comparison with intraperitoneal injection,oral delivery of peptide PW5 altered the gut microbial ?-and?-diversities,and reversed the relative abundance of ten differential genera,which werehighly correlated with disease progression.The gut microbiota composition after oral administration of PW5 was more closely to wild-type mice.This study demonstrated that walnut protein had great efficacy for improving learning and memory impairments,and confirmed that oral administration of walnut protein-derived bioactive peptide PW5 could significantly ameliorate cognitive impairments better than intraperitoneal injection.These findings not only can broaden the potential application scope of walnut protein to improve its economic,nutrition and medical values,but also suggested that walnut protein-derived bioactive peptides could be further developed into functional foods or drugs with learning and memory-improving properties,which can be applied in the prevention,management and treatment of various diseases related to cognitive impairments.