Study On The Effect Of Pine Nuts Protein Regulating Nrf2 Signaling Pathway In Protecting Neurons And Its Mechanism

Objective:To investigate the role and mechanism of pine nut protein protection neurons.Methods:（1）The pine nuts protein was extracted by salting out method,the molecular weight range of pine nuts protein was detected by SDS-PAGE,and the amino acid composition and content of pine nuts protein were analyzed by automatic amino acid analyzer.（2）In vitro experiment:Using SH-SY5Y cells treated with hydrogen peroxide,a model of cellular oxidative damage was established.The cells were divided into Blank group,Model group,Pine nuts protein low-dose group,Pine nuts protein medium-dose group,and Pine nuts protein high-dose group.The effect of pine nuts protein on the survival rate of SH-SY5Y cells was detected by MTT assay.The effect of pine nuts protein on the apoptosis of SH-SY5Y cells was detected by flow cytometry.The expression levels of Bcl-2,Bax,Nrf2,HO-1,Keap1 and Aβ_1-42proteins in SH-SY5Y cells were detected by Western blotting.RT-PCR technique was used to detect the expression levels of Bcl-2,Bax,Nrf2,HO-1,Keap1 genes in neural cells.（3）In vivo experiment:ICR mice were selected and used in Vascular dementia model mice,which were divided into Sham group,Model group,Positive drug group,Pine nuts protein low-dose group,Pine nuts protein medium-dose,and pine nuts protein high-dose group.These groups were used to detect the behavior of mice by Morris water maze and platform test.The effects of pine nuts protein on the antioxidant capacity of mice hippocampus were detected by SOD,MDA and GSH biochemical kits.The effects of pine nuts protein on the morphology of mice hippocampal neurons were observed by HE staining.TUNEL staining was used to detect the effect of pine nuts protein on apoptosis of mouse hippocampal neurons.Western blotting was used to detect expression levels of Bcl-2,Bax,Nrf2,HO-1,Keap1,and Aβ_1-42-42 proteins in mice brain.Results:（1）Pine nuts protein is mainly distributed in 3.3-14.4 KDa and20.1-25KDa.Pine nuts protein contains 7 essential amino acids and 10 non-essential amino acids.（2）Pine nuts protein could increase the survival rate of SH-SY5Y cells induced by hydrogen peroxide（P<0.05）,and inhibit oxidative stress and apoptosis of SH-SY5Y cells（P<0.05）.（3）Pine nuts protein can reduce the escape latency and escape route of vascular dementia model mice（P<0.05）.Pine nuts protein can maintain the integrity and alignment of mouse hippocampal neurons,inhibit nuclear pyknosis,and reduce hippocampus neurons apoptosis in mice.Pine nuts protein can increase the levels of GSH and SOD in brain tissue（P<0.05）,and decrease the content of MDA in brain tissue（P<0.05）.Both in vitro and in vivo experiments showed that pine nuts protein can up-regulate the expression levels of Nrf2 HO-1 and Bcl-2proteins（P<0.05）in nerve cells,and down-regulated the expression levels of Aβ_1-42,Keap1 and Bax proteins in nerve cells（P<0.05）.Conclusion:Pine nuts protein may up-regulate the expression levels of Nrf2,HO-1 and Bcl-2 by promoting the Nrf2/HO-1 signaling pathway,down-regulate the expression levels of Aβ_1-42,Keap1 and Bax proteins,which could inhibit oxidative damage and the apoptosis of neuronal cells.Therefore,pine nuts protein produces neuronal protection.