Study On Improving Sleep Deprivation-induced Memory Impairment In Rat By Walnut (Juglans Regia) Peptides And Its Neuroprotective Mechanisms In PC12 Cells

Impairment of learning and memory is widely considered as the main clinical syndrome in many neurodegenerative diseases.Loss of sleep is one of the main reasons for some neurodegenerative diseases associated with memory deficits in modern society.Therefore,it is of great importance to develop functional foods to alleviate memory deficits associated with loss of sleep.Walnut peptides have been demonstrated to show neuroprotective effects in various animal models.However,whether walnut peptides could ameliorate the sleep deprivation?SD?induced learning and memory impairment in rat is still unknown.Additionally,previous studies mainly focused on the evaluation of neuroprotective effects of WPH.However,the structure characteristics of neuroprotective peptides in WPH are still unknown.Therefore,the aim of this study was to determine the neuroprotective effects of walnut protein hydrolysates?WPH?against memory deficits induced by SD in rat and further to identify and characterize the potent neuroprotective peptides against glutamate-induced apoptosis in PC12 cells.A remarkable amelioration effect on behavioral performance in Morris water maze test was observed for WPH and its low molecular weight fraction WPHL,especially for WPHL.Additionally,a reduction of antioxidant defence?CAT,GSH-px,and SOD?and GSH/GSSG as well as an increase of MDA content induced by SD were normalized in brain of rat after administration of WPH and WPHL.It indicated that walnut peptides could attenuate SD-induced memory deficits via alleviating oxidative stress.In conclusion,WPHL showed a stronger neuroprotective effect than WPH.WPHL showed stronger learning and memory improvement capacity than WPH in vivo.Therefore,WPHL was chosen for further purification of potential neuroprotective peptides.WPHL was separated into six fractions?G1-G6?by Sephadex G-15 gel filtration chromatography.G4 and G6 exhibited the strongest neuroprotective effects on glutamate-treated PC12 cells among all fractions.Therefore,G4 and G6 were further analyzed by UPLC-MS/MS.A total of 18 peptides were identified from WPHL by combination of searching Mascot and manual de novo.Among them,GGW,VYY and LLPF exhibited protective effects on glutamate-induced apoptosis in PC12 cells.Thus,these three peptides were chosen to further study the underlying molecular mechanisms of protective effects mediated by oxidative stress and related apoptosis pathways in PC12 cells.GGW,VYY and LLPF could protect PC12 cells against glutamate-induced apoptosis and reduce ROS production in PC12 cells.The possible mechanism underlying their protective effects of GGW and VYY could be related to their strong radical scavenging activity as well as their ability to reduce the depletion of SOD and GSH-px in PC12 cells.Notably,the marked neuroprotective effects of LLPF,which did not show obvious free radical scavenging activity in vitro,could be attributed to its strong effects on inhibiting Ca²⁺influx and MMP collapse.Additionally,all these peptides could regulate the expression of apoptosis-related proteins?Bax and Bcl-2?.Therefore,walnut peptides might be regarded as the potential nutraceuticals against neurodegenerative disorders associated with memory deficits.