Screening Of Bioactive Peptide From Oysters And Its Mechanism Of Ameliorating Cognitive Impairment Of Aged-zebrafish

With the aging of the global population,the number of patients with cognitive impairment in the elderly is increasing,which has become a problem that can not be ignored in the field of public health security.Therefore,it is of great social significance to study the functional components and mechanism of improving cognitive impairment.Previous studies have confirmed that oyster hydrolysates can improve the learning and memory ability in mice.The research,however,has not yet been systematically and extensively performed and little information on ameliorating cognitive impairment of oysters by using the natural aging animal model was reported.In this research,hydrolysates from enzymatic hydrolysis oysters(Crassostrea hongkongensis)protein and the natural aging zebrafish were selected as the animal model to screening active.And explore the effect of active ingredients on cognitive impairment of elderly zebrafish.The component purified with using various methods,including ion-exchange chromatography,G25 gel filtration,semipreparative chromatography.The amino acid sequence of biological peptides was identified by ESI-MS/MS and synthesized by artificial methods.Finally,cell experiments were used to explore the molecular mechanism of synthetic peptides in improving cognitive impairment.The research provides a theoretical basis for the development of oyster functional foods in improving cognitive impairment.The main contents and results of this dissertation are as follows:(1)The escape latency in fading step-through tests was notably prolonged,the escape distance and latency were significantly decreased in Morris water maze,and the crossing time in spatial probe tests was significantly increased for the mice treated with the high-dose oyster hydrolysate.The results showed that high-dose oyster hydrolysate could significantly improve the learning and memory of normal mice than low-and medium doses.(2)Ultrafiltration(UF)2(5-8 k Da)exhibited the most significant improvement in cognitive impairment of the aged zebrafish compared to other components(UF1: 8k Da,UF3: 5-3 k Da,and UF4: < 3 k Da).It enhanced the success rate of zebrafish entering the food storage area on the fourth day in behavior tests,decreased latency,and prolonged the swimming distance and time on the second,third,and fourth day.In the determination of amino acid components of each ultrafiltration components the found that aromatic amino acids,hydrophobic amino acid,glutamic acid,and aspartic acid in UF2 were much higher than those in the other three components.In addition,in the antioxidant activity,anti-inflammatory and neurotransmitter content,it can be demonstrated that UF2 has significant hydroxyl radicals and superoxide radicals scavenging activities,the levels of SOD and CAT increased significantly,the activity of BV2 cells induced by LPS decreased,and concentrations of Ach and r-GABA increased.These results suggested that the ameliorative effect of UF2 on cognitive impairment in aged zebrafish was related to the improvement of antioxidant,anti-inflammatory activities and the up-regulation of neurotransmitter secretion.(3)Behavioral tests show that the cognitive ability of aged fish is lower than younger ones.UF2 could significantly ameliorate cognitive impairment of aged zebrafish with significant increases in success rate,swimming distance,and time in the right arm and a decrease in latency.Analysis results of biochemical and gene expression show that UF2 increased the activities of SOD and CAT but decreased MDA,IL-1β,TNF-α,and the gene levels of cd11 b and sabg.It could upregulate the levels of gfap,psd-95,bdnf,and trkb and increase the neurotransmitters(Ach and r-GABA)and BDNF.It also increased the numbers of neurons and ameliorated apoptosis.The effects might be associated with the regulation of glia cell function,reduction of oxidant stress,and inflammation and thereby leading to neuronal protection.Furthermore,UF2 upregulated the levels of bdnf,trkb,and neurotransmitters and enhanced synaptic function,resulting in the recovery of ageing-induced memory deficits..(4)Fourteen bioactive peptides were identified with mass spectrometry after isolated.It was confirmed by using the molecular docking method that the peptide5(IDYER)and peptide18(IAFIMDESNVLDSGFLER)possessed better activity to activate CREB.(5)Cell experiments showed that the synthesized peptides DYER and IAFIMDESNVLDSGFLER significantly inhibited the production of intracellular ROS and NO in LPS-induced BV2 cells,effectively intervened in cell aging in D-gal-induced SH-SY5 Y cell.They also could inhibit the phosphorylation of nuclear transcription factor NF-κB(p65)and degradation of IκB,increase the phosphorylation of ERK1/2 and CREB,down-regulate the level of BAX,and up-regulate the level of BCL-2.The results revealed that the synthesized peptides can activate the ERK-CREB signaling pathway via inhibiting the NF-k B/Ik B signaling pathway and apoptosis signal pathway thereby recovering the aging-induced memory deficits.