The Mechanism Of Walnut Peptide Protecting Glial Cells Based On Inhibition Of Oxidative Stress And Inflammation

Walnut peptides have anti-inflammatory and antioxidant biological activities.This study is based on the preliminary screening of walnut peptides EVSGPGLSPN,WEKPPVSH,LPLLR with strong antioxidant activity in the early stage of the laboratory.Using oxidative damage model and cell inflammation model to study its protective effect on primary astrocytes and microglia The specific results are as follows:1.By collecting mouse cerebral cortex,after shredding,trypsin and DNase digestion,differential adherence purification,serum-free starvation treatment,constant temperature shaker purification,to obtain mouse primary astrocytes,immunofluorescence Up to 95% positive cells can be identified.2.Treat mouse astrocytes with 400μM H₂O₂ to establish an oxidative damage model and investigate the relief effect of walnut peptide on oxidative stress.The experimental results show that the walnut peptides EVSGPGLSPN,WEKPPVSH and LPLLR can reduce the ROS content in the cells（P <0.01）,at 50 μM to 69.36%,39.78% and 58.51% of the model group,respectively,and significantly increase the total antioxidant Levels and the activities of superoxide dismutase（SOD）and catalase（CAT）（P <0.01）.3.Stimulate BV2 cells by LPS,establish a BV2 inflammation model,and examine the inhibitory effect of walnut peptides on inflammatory factors.The experimental results show that the walnut peptides EVSGPGLSPN,WEKPPVSH and LPLLR have a good protective effect on inflammatory injured BV2 cells,of which WEKPPVSH activity is the highest,when the concentration reaches 50 μM,the NO secretion is reduced to 41.2% of the model group.The secretion of IL-1β,IL-6,and TNF-α were significantly reduced（P <0.01）,and fell to 34.32%,63.43%,and 40.41% of the model group,respectively.4.To study the effect of WEKPPVSH on key proteins in NF-κB signaling pathway of BV2 cells by immunofluorescence and Western blot.The results of immunofluorescence experiments show that WEKPPVSH can reduce the nuclear transport of p65 protein;Western blot experiments show that WEKPPVSH down-regulates the expression of downstream inflammatory factors iNOS and COX-2 by inhibiting the phosphorylation of IκB-α and p65 protein.5.Analysis of the effect of WEKPPVSH on key proteins in the Nrf2 signaling pathway of BV2 cells using Western blot.The results showed that WEKPPVSH significantly up-regulated the expression of Nrf2 protein（P <0.05）,and further significantly up-regulated the activities of downstream antioxidant enzymes HO-1,SOD and CAT（P <0.01）,and reduced intracellular ROS levels（P <0.01）.6.Determine the stability of walnut peptides by high performance liquid chromatography.The experimental results prove that the walnut peptide EVSGPGLSPN has good pH stability and digestion enzyme tolerance,poor stability to high temperature,and begins to degrade at 80 °C;WEKPPVSH has good thermal stability and pH stability,and has a high pepsin Tolerated,but hydrolyzed by trypsin;LPLLR has good thermal stability,pH stability and resistance to digestive enzymes.