Protective Effects Of Walnut Peptides On Alzheimer’s Disease Model In Vivo And Vitro

This paper launched the following two projects: In projectⅠ, we explore the protective effects of peptides released from walnut（Juglans Sigilata Dode） on hydrogen peroxide and amyloid beta（25-35）-induced injury in rat pheochromocytoma cells and its possible mechanisms. In project II,the safety of walnut peptides was explored by acute toxicity and subacute toxicity study, and then the activity of anti-Alzheimer’s disease（AD） and the potential mechanism were also explored. Project Ⅰ:Objective: To investigate the protective effect of walnut peptides on the injury of PC12 cell induced by the β-amyloid protein（Aβ25-35） and H₂O₂. Methods: Walnut peptides were administrated after the PC12 cells were injury by the H₂O₂ and Aβ25-35, the cell viability were measured by the means of MTT. The levels of MDA and the activity of SOD, GSH-P_X and CAT were detected by the reagent kits. To further investigate the machine of the neuroprotective effect of walnut peptide, the intracellular ROS was monitored by using the DCFH-DA fluorescent probe Results: Compared with the model group, the walnut peptide treatment groups significantly increased the viability of the cell induced by the H₂O₂ and Aβ25-35. The data also shows that pre-incubation with walnut peptides remarkably decrease the LDH leakage and significantly decrease the content of MDA and increase the activity of SOD, GSH-P_X and CAT in the PC12 cell system. Conclusion: Walnut peptides have effective protection against H₂O₂- and Aβ25-35-induced injury of PC12 cell, which may be related to its anti-oxidant action.Project II: Objective: The present study aimed to determine the protective effects of walnut peptides against the neurotoxicity induced by Aβ25-35 in vivo. Methods: Aβ25-35 was injected into bilateral hippocampi of mice to induce AD model. Mice were treated with distilled water or walnut peptides（200 mg/kg/day, 400 mg/kg/day and 800 mg/kg/day, p.o.） for five consecutive weeks. Spatial learning and memory of mice were investigated by the Morris water maze test and step-down avoidance test. To further explore the underlying mechanisms of the neuroprotectivity of walnut peptides, the activities of superoxide dismutase（SOD）, glutathione（GSH）, acetylcholine esterase（ACh E）, and content of malondialdehyde（MDA） and level of nitric oxide（NO） in the hippocampus of mice were measured by spectrophotometric method. Furthermore, the levels of 8-hydroxy-2’-deoxyguanosine（8-OHd G）, tumor necrosis factor-α（TNF-α）, interleukin 1β（IL-1β） and interleukin 6（IL-6） in the samples were determined using ELISA and hippocampal expressions of inducible nitric oxide synthase（i NOS） and nuclear factor B（NF-κB） were evaluated by western blot analysis. Results: Data from this study showed that walnut peptides supplementation effectively ameliorated the cognitive deficits and memory impairment of mice. Meanwhile, our study also revealed effective restoration of levels of antioxidant enzymes as well as inflammatory mediators with supplementation of walnut peptides（400 or 800 mg/kg）. Conclusion: All the above findings suggested that walnut peptides may have a protective effect against AD via reducing inflammatory responses and modulating antioxidant system.