Wheat Germ Peptide?RVF? Attenuate Hydrogen Peroxide?H₂O₂?-induced Apoptosis And Oxidative Stress Via The NF-?B Pathway In SH-SY5Y Cell Lines

Active radicals oxygen species?ROS?,generated by cell metabolism,play an important role in the cell signaling,apoptosis,gene expression and ion transport.Over-generated ROS in cells tend to attack acid side chains of nucleic acid,protein and amino,as well as the double chains of adverse reaction of unsaturated fatty acids,and this causes the pathological process of oxidative damage of the cells.Current research has confirmed that the wheat germ peptides have some antioxidant property,but most of them are limited to use chemical method in vitro test or simple animal experiments in vivo.However the comprehensive analyses of the antioxidant peptides system research in cells combine with the study of the molecular mechanisms in the body was rarely reported.In the previous study,we have used chemical method and cell experiment tested the antioxidant mechanism of the wheat germ peptide RVF?Arg-Val-Phe?,and antioxidant property of it was found.In the present study,we will further explore the nutrition function and antioxidant mechanism of RVF.To explore its comprehensive fouction,the model of staration damage and H₂O₂-induced oxidative stress in human neuroblastoma cell line?SH-SY5Y cells?was used to investigate the nutrition function and the neuroprotective effects.We found that RVF provided important basis in reducing oxidative stress,prevention of neurodegenerative diseases,also has provided the new mentality for the comprehensive utilization of wheat germ protein.RVF,peptide of wheat germ obtained by separation and purification,then compound by solid phase,was chosen as the antioxidant used in the model that SH-SY5Y cells starved or treated with H₂O₂,then explore the protection effect of starved and oxidative damage from cell survival rate and morphology.However,in the model of starvation,RVF had no obvious nutrition protection,while pre-incubation with the 200?mol/L RVF for 12 h could increase cell survival rate by 25.05%and also observed the shrinkage cell recover long and thin spindle,living cells volume also increased significantly in morphology aspect in a dose-dependent manner.The study explored O₂^-·level by flow cytometry and superoxide dismutase?SOD?,the ratio of GSH/GSSG and the protein carbonyl was measured by colorimetry.As the result showed,the O₂^-·and the protein carbonyl level of group pretreatment RVF had a significant increase by 30.09%and 2.03 nmol/mg protein compared with the H₂O₂ group,respectively.While the cellular SOD and the ratio of GSH/GSSG level of group treated with 100,150,200?mol/L wheat germ antioxidant peptide RVF,compared with H₂O₂ group,increased 37.72%and 2.987 respectively.The study investigated the DNA damage by TUNEL/hochest 33342 double staining.RVF could successfully reduce the damage by 24.47%.Apoptosis was also assessed by detecting apoptotic protein through performed electrophoretic mobility shift assay?EMSA?,RT-PCR and western blot method.Firstly,H₂O₂ resulted in I?B?protein decreased and overexpressed of pI?B?^Ser32 after 30 min of exposure,then indicating the induction of p65nuclear translocation reaching a maximum effect at 60 min,an essential step in NF-?B activation,and promote the NF-?B DNA binding activity,another step in NF-?B activation.RVF?50,100,200?M?reversed the change on H₂O₂-induced cells with a dose dependent in the decreased I?B?protein and increased of pI?B?^Ser32,and also retarded H₂O₂-induced the induction of p65 nuclear translocation.Meanwhile,there were less detectable DNA-protein complexes using nuclear proteins extracted from cells pretreated with 200?M of RVF.These results indicate that RVF protected H₂O₂-induced the activation of NF-?B in SH-SY5Y cells by a redox-sensitive mechanism.It was also shown that RVF reduced activation of p53 at1.76 times by NF-?B is essential for H₂O₂-induced apoptosis in SH-SY5Y cell lines,then decrease the Bax protein,which is the mitochondrial apoptotic pathways related proteins,downstream proteins of p53 protein.RVF could also efficiently enhance the activation of XIAP and cFLIP m RNA through RT-PCR method.Furthermore,RVF inhibited H₂O₂-induced cleavage of Caspase-3,8,9 known as the active form.In summary,our study showed that antioxidant peptides of wheat germ have no obvious nutrition protection in the model of starvation,but have a significant protective effect in SH-SY5Y cells against H₂O₂-induced oxidative injury.In Conlusion,in one hand,the inhibition of O₂^-·level,SOD,the ratio of GSH/GSSG and protein carbonyl,and the prevention the activation of the NF-?B apoptosis pathway contributed to the protective mechanisms.In the other hand,the increasing the expression of anti-apoptotic proteins and decreasing the expression of pro-apoptotic proteins also played an important role in the protection.