Mechanism Of Walnut-derived Antioxidant Peptides In Improving Learning And Memory Dysfunction

With the acceleration of global population aging,China is also facing drastic changes in m TORpopulation structure.By the end of 2020,the population aged 60 years old and above in China has reached 260 million,accounting for 18.7%of the population,which is expected to increase to 37.2%by 2050.There is a serious increasing situation for public health challenges brought by the aging population.In particular,the decline of cognitive function in the elderly causes huge emotional burden and economic pressure to the family and society.Studies have shown that cognitive function is plastic,and appropriate interventions can effectively protect cognitive function or delay its decline,among which dietary intervention is one of the effective means.Therefore,it is of great significance to find green,safe,efficient and neuroprotective functional factors for dietary intervention to prevent cognitive decline.In China,there are abundant walnut resources,especially in Changbai Mountain area of high yield of wild walnut,and good quality.Walnut proteins enzymatic hydrolysate(<3 k Da fraction)has been proved to have the activity of improving learning and memory in mice,but the structure and molecular mechanism of the peptide remain to be unclear.Therefore,the purified and identified walnut peptides in this paper were used as the research object,and BIOPEP database was used to screen walnut peptides with potential memory improvement and antioxidant activity.Through in vitro experiments,walnut peptides with high antioxidant activity were screened out.Combined with autophagy activator and autophagy inhibitor,the protective mechanism of walnut peptide on oxidative stress induced by A?_25-35 in nerve cells was investigated.The neuroprotective effect of walnut peptide on improving learning and memory impairment in mice was verified by animal experiment in vivo.Based on proteomics and phosphorylomics,the biological function of walnut peptide in relieving scopolamine-induced learning and memory impairment in mice was determined.Combined with NRF2 inhibitor,it further revealed that walnut peptide can protect HT-22 cells from oxidative damage induced by H₂O₂,and elucidated the mechanism of walnut peptide in improving nerve damage and learning and memory impairment.The main results are as follows:(1)Structural identification and screening of walnut peptides.The structure of purified peptides was identified by de novo sequencing,and 645 walnut peptides were selected from2341 peptide sequences with confidence?90%as the index.According to the structural characteristics of memory peptides reported in BIOPEP database(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep)and literatures,12 peptides with relatively high content of hydrophobic amino acids and aromatic amino acids and unreported dual effects of improving memory and antioxidation were selected.Using chemical experiment ORAC,combined with PC12 cell model in vitro,taking ROS,apoptosis,ATP and GSH-Px as indicators,3 walnut peptides with high antioxidant activity were screened.RP-HPLC was used to detect the stability of walnut peptides with high antioxidant activity.The experimental results showed that TWLPLPR(20?-60?),YVLLPSPK(20?-80?)and KVPPLLY(20?-100?)have high temperature resistance characteristics,and walnut peptides with high antioxidant activity can not be degraded after pepsin and trypsin digestion,which lays a foundation for the following research.(2)Preliminary the mechanism of walnut peptides TWLPLPR,YVLLPSPK and KVPPLLY promoting autophagy and improving oxidative stress.Taking PC12 cells induced by A?_25-35 as neurotoxicity model,combined with autophagy activator RAPA and autophagy inhibitor 3-MA,it was proved that walnut peptides activated the Akt/mTOR signaling pathway by significantly regulating the protein expressions of p-Akt/Akt(p<0.01)and p-mTOR/mTOR(p<0.01).Promoting autophagy related protein(LC3?/LC3?r atio,p62,Beclin 1)and lysosome protein(LAMP1,LAMP2,Cathepsin D)expression,increase cytolysosome and autophagy-lysosome quantity,thus significantly reduced A?_25-35 induced PC12 cells ROS levels(p<0.01)and the level of cell apoptosis,significant increase in the activity of ATP(p<0.01).Walnut peptides TWLPLPR,YVLLPSPK and KVPPLLY mediated autophagy through Akt/mTOR signaling pathway to improve A?_25-35-induced oxidative stress in PC12 neurons,thus providing experimental and theoretical basis for the study of protecting neurons from oxidative damage.(3)Study on improvement of learning and memory impairment in mice by walnut peptide YVLLPSPK.The scopolamine-induced aging mice were used as the experimental animal model.The intragastric administration of walnut peptide,YVLLPSPK,did not cause other side effects,nor did it cause abnormal changes in hair,food intake,water intake,and behavior of C57BL/6J mice,and the weight of mice did not change significantly(p>0.05).Using H&E staining,the tissue structure of mice was observed in detail under different multiples.The experimental results showed that walnut peptide did not cause inflammation,necrosis,degeneration,hyperplasia and fibrosis in the liver and kidney of mice,which proved that walnut peptides had no toxic and side effects on mice.Morris water maze behavioral experiment was used to study the effect of walnut peptide on the improvement of learning and memory in mice induced by scopolamine.The experimental results showed that in the place navigation trial,walnut peptide significantly reduced the escape latency(p<0.05)and movement distance(p<0.01)of mice,and significantly increased the movement distance(p<0.01)and time(p<0.01)of mice in the effective area.In the spatial probe trial,the peptide significantly increased the times of passing the original platform place(p<0.01),the time of detention platform(p<0.01),and the movement distance and time of in the effective area(p<0.01).Rhodamine B-labeled peptide YVLLPSPK was used for in vivo imaging detection of mice.The experimental results showed that walnut peptide can be absorbed by blood and distributed in various organs of the body(kidney,liver,spleen,bladder,large intestine and small intestine)with time.The above experiments indicated that walnut peptide can maintain its integrity after oral administration,and then be absorbed and utilized by the body to improve the learning and memory impairment of mice induced by scopolamine.It has potential development and application value in plant-derived functional foods,and provides a feasibility basis for its application in functional foods or medicine fields in the future.(4)Proteomics analysis of the effect of walnut peptide YVLLPSPK on scopolamine-induced hippocampal tissue in mice.Total protein and phosphorylated 4D-label free quantitative proteomics of mouse hippocampus were performed to analyze the effect of walnut peptide YVLLPSPK on scopolamine-induced differential protein expression in the hippocampus of C57BL/6J mice.Through GO and KEGG enrichment and cluster analysis of differential proteins(fold change>1.5),differential proteins are mainly concentrated in biological processes such as transcription and translation,mitochondrial phospholipid transport,microtubule movement regulation,membrane transport,neuron regeneration,differentiation and movement,etc.They are related to the components of membranous organelles and membranes in synapses,and are mainly related to molecular functions such as transmembrane transporters,VDAC and ATPase activities.The results of the KEGG pathway showed that differential proteins were enriched in transport,catabolism and cell movement pathways.Cluster enrichment results showed that walnut peptide improved learning and memory impairment induced by scopolamine in mice,which may be related to mitochondrial biological function pathway and participate in regulating membrane systems containing different transporters.(5)The mechanism of walnut peptide YVLLPSPK mediates PINK1 to promote mitochondrial autophagy and improve neuron impairment.The walnut peptide YVLLPSPK significantly reduced the levels of protein carbonyl(p<0.01),LPO(p<0.01),8-OHd G(p<0.01)and TUNEL apoptosis(p<0.01),and significantly increased ATP activity(p<0.01)induced by scopolamine in mouse hippocampus.The walnut peptide restored the morphology of mitochondria in the hippocampus of mice induced by scopolamine,reduced mitochondrial vacuolation and increased mitochondrial cristae.Western blot experiment showed that the walnut peptide significantly increased the expression of mitochondrial fusion protein(MFN2)in mouse hippocampus induced by scopolamine(p<0.05),and significantly decreased the expression of mitochondrial division protein(DRP1)(p<0.01),VDAC1(p<0.01)and CYCS(p<0.01).The walnut peptide significantly increased the expression level of mitochondrial outer membrane markers PINK1and Parkin protein(p<0.01).The ratio of autophagy marker LC3-II/LC3-I and Beclin-1 protein were significantly increased(p<0.01),the expression of p62 protein was significantly decreased(p<0.01),and the expression level of lysosomal related protein 1(LAMP1)was significantly increased(p<0.01).The results of immunofluorescence assay showed that the walnut peptide significantly increased the co-localization of autophagy marker LC3-II and mitochondrial marker protein PINK1 in mouse hippocampus induced by scopolamine(p<0.01).The walnut peptide significantly increased the expression of NRF2 and HO-1 protein in the nucleus of mouse hippocampus induced by scopolamine(p<0.01),and significantly decreased the expression of KEAP1 protein(p<0.01).The results of immunofluorescence assay also showed that the walnut peptide significantly increased the translocation of nuclear NRF2 in the mouse hippocampus induced by scopolamine(p<0.01).At the same time,the inhibitor ML385significantly regulated the effects of walnut peptide on mitochondrial related proteins(DRP1?MFN2?VDAC1?CYCS?PINK1?Parkin)and mitochondrial autophagy related proteins(LC3-II/LC3-I ratio?Beclin-1?p62?LAMP1)in HT-22 cells induced by H₂O₂(p<0.01).Flow cytometry showed that walnut peptide significantly reduced the levels of ROS,apoptosis and Mito SOX in HT-22 cells induced by H₂O₂(p<0.01).Observed by fluorescence microscopy,the walnut peptide YVLLPSPK significantly increased the level of MMP(p<0.01).However,after treatment inhibitor ML385,intracellular ROS,apoptosis,mitochondrial superoxide and MMP levels were significantly inhibited(p<0.01).The above experiments provide theoretical support and experimental basis for walnut peptide to improve learning and memory impairment,and also lay a foundation for developing food-borne active substances to improve learning and memory function in the future.