Study On Mechanism Of Memory Improvement By Synergistic Action Of Shrimp Peptide And Maize Peptide Through Scopolamine-induced Amnesiac Mice

Natural foodborne bioactive peptides are small molecule active substances with high nutritional value and physiological activity.It comes from a wide range of sources,simple preparation process,non-toxic side effects,and can be quickly absorbed by the human body.The development of specific physiological activities of active peptides has gradually become a research hotspot.The society in today is under great pressure and the fast pace of life.The people have memory amnesia and forgetfulness.However,there are few studies on improve memory ability of bioactive peptides,and evaluation as well as bioactivity mechanism.In this study,bioactive peptides with high antioxidant activity were obtained by hydrolysis shrimp protein and specific methods of isolation,purification and identification.It was explored the mechanism of shrimp peptide that improve ability of inhibit scopolamine induced oxidative stress injury and neuroprotective ability in PC12 cells.The shrimp peptide with the best protective ability was selected out for animal model verification.The synergistic effect of shrimp peptide and maize peptide was screened out to enhance the neuroprotective ability and study its mechanism.Then,it was investigated that the effect of peptide scopolamine induced memory impairment in mice.This study aimed to develop new functional food,provided theoretical and methodological guidance for improving the mechanism of memory ability and its application in industrial production.The main results of research were as follows:（1）Using shrimp protein surimi as raw material,four different single factor experiments and response surface test were conducted to optimize experiment.The DPPH free radical scavenging of hydrolysis production was taken as the index to obtain the optimal technological conditions:pH value 9.0,temperature 60℃,enzyme content 6.09%,substrate concentration1.76%.The actual DPPH free radical scavenging rate was 28.15±0.97%.Ultrafiltration was used to seperate the shrimp protease hydrolysates into four components,among which SPH_{<1 k Da} had the highest antioxidant capacity（P<0.05）.The SPH_{<1 k Da} was purified by Sephadex G-25 and obtained four groups.F1 and F2 had the highest DPPH and hydroxyl radical scavenging activities.F1 and F2 were identified by ultra-high liquid chromatography-mass spectrometer and the sequences was KMDDK,KMDDQ,QMDDK,QMDDQ,MTTNI and MTTNL.（2）The structure-activity relationship of PC12 cell model for memory enhancing shrimp peptide was investigated.The polypeptide QMDDQ and MTTNL had the strongest antioxidant capacity by EPR.The optimal induction conditions of scopolamine induced cell damage model were established.The cell survival rate was 49.23±0.4%when treated with scopolamine 4mg/mL for 4 h.The concentration of 0.5μg/mL-1.0 mg/mL shrimp peptides treated PC12 cells without toxicity or proliferation.Secondly,QMDDQ,KMDDQ,MTTNI and MTTNL significantly increased SOD activity and reduced ROS content（P<0.05）.The shrimp peptides could improve cell status and inhibit cell apoptosis.In addition,AChE activity was significantly decreased,and ACh content was increased（P<0.05）.The fluorescence intensity of p-CREB were reduced（P<0.05）.Finally,¹H NMR spectrum and COSY-NOSEY spectrum showed that the amino acid composition,location and spatial structure of QMDDQ and MTTNL were different from other blue shrimp protein peptides,which made the peptides extend in the spatial structure and then combine with external substances,thus possessing stronger activity ability.（3）The mechanism of targeted regulation of memory enhancing assisted by shrimp peptide was investigated.The shrimp peptides QMDDQ,KMDDQ,MTTNL and MTTNI were significantly increased the protein expression of BCl-XL,and decreased the protein expression of Bax and Caspase-3（P<0.05）,so as to participated in the apoptotic protein-mediated cascade reaction and inhibited cell apoptosis.On the other hand,shrimp peptides significantly increased the protein expression of p-PKA,p-CREB/CREB,BNDF and NGF（P<0.05）,activated the neural signaling pathway PKA-CREB-BNDF,and inhibited nerve cell injury.Animal model experiments were conducted to verify that the shrimp peptide QMDDQ could improve the memory impairment in mice.The mechanism of QMDDQ was mainly through acetylation,succinylation and lactate modification to change the key enzymes and proteins in the nerve cell metabolic pathway,and then improved the memory loss and cognitive impairment in mice.（4）Mechanism of synergistic regulation of shrimp peptide-maize peptide based on PC12cell model to enhance memory peptide was investigated.The best ratio and concentration were selected by EPR combining QMDDQ with maize peptides HALGA and AGLPM.QMDDQ+AGLPM could improved the disturbance of energy metabolism caused by scopolamine injury in varying degrees,and activated pathway of energy metabolism.QMDDQ+AGLPM increased the expression of anti-apoptotic protein BCL-XL,and decreased the expression of pro-apoptotic proteins Bax,Caspase-3 and p53（P<0.05）.QMDDQ+AGLPM significantly reduced the intracellular AChE activity and increased ACh content（P<0.05）.It inhibited the apoptosis of nerve cells,blocked the expression and transmission of apoptotic signals.QMDDQ+AGLPM significantly increased the fluorescence intensity of SynGAP,and increased the protein expression levels of synapse proteins p-Ca MKII,SynGAP,PSD95 and p-CREB（P<0.05）.It analyzed the synergistic activation of mitochondrial energy metabolism,then inhibited the apoptosis and activated synaptic plasticity signal transduction pathways to improve nerve cell injury.（5）Based on animal experiment,the synergistic protective effect of shrimp peptide QMDDQ and maize AGLPM on memory impairment induced by scopolamine was investigated.The average daily intake,drinking water,body weight change,body composition analysis and MRI showed that the gavage polypeptide had no obvious effect on the physiological condition of the mice.Behavioral experiments showed that the shrimp-maize peptide could effectively improve the memory impairment caused by scopolamine in water maze,dark escape experiment and shuttle box experiment,and the improvement effect was better than that of shrimp peptide or maize peptide alone.The AChE activity was significantly decreased,and ACh content and SOD activity were significantly increased in the shrimp-maize peptide group.HE staining results showed that the peptide groups had clear and complete morphology of the CA1 region,CA3 region and DG region in the hippocampal.The small number of nerve cells showed shrinkage.The nucleus and cytoplasm of peripheral nerve cells were decreased,but most of them were orderly arranged.The hippocampal damage was alleviated,which was better than the scopolamine group.HE staining of liver,kidney and colon in mice had no pathological changes.The results showed that the shrimp-maize peptide could improve the damage and metabolic disorders induced by scopolamine,and then improve the memory loss and cognitive impairment in mice.