The Anti-oxidative Effects And Mechanism Of Antioxidant Peptides From Egg White Against Oxidative Stress Injury In Human Embryonic Kidney293Cells

This research is mainly engaged in oxidative stress inhibitory activity of eggwhite peptides. This study takes the pepsin hydrolysates from egg white ovomucin asraw materials, and pentapeptides from egg white as research object.First of all, in order to evaluate the antioxidant activity of pentapeptides from eggwhite protein, we adopted chemical methods based on HAT and HET mechanisms,respectively. The pentapeptide CFDVF showed extraordinary DPPH scavengingactivity (50.14±2.1%at the concentration of1.0mM), VYQFL exhibited thestrongest ABTS free radical scavenging ability (TEAC value=94.66±0.37μMTE/100μM), WNWAD showed extraordinary oxygen radical absorption capacity(ORAC value=2.53±0.18μM TE/μM). The antioxidant activity of pentapeptidesmay be attributed to the absence of Trp、Tyr and Cys residue, which can serve asproton and hydrogen donors.Secondly, in order to further examine the antioxidant activity of pentapeptidesfrom egg white, the oxidative stress model of HEK293cells was established by MTSassay. The optimal injury concentration of H2O2to establish the oxidatively damagedHEK293cell model was400μM, and the optimal time to treat the cells was24h,when the HEK293cell viability reduced to approximately52.6±2.4%of the controlvalue. Through this model, WNWAD (Trp-Asn-Trp-Ala-Asp) exhibited the strongestoxidative stress inhibitory activity. The pentapeptides showed no stimulatory orcytotoxic effects in HEK293cells.Finally, the WNWAD was screened out to carry out the research on mechanismof pentapeptides from egg white against oxidative stress in human embryonic kidney293cells. The oxygen radical absorption capacity, cellularly antioxidant capacity andcytotoxic property of low doses of WNWAD were evaluated. Furthermore, themorphology and surface microstructure of HEK293cells were observed by ScanningElectron Microscope. The intracellular ROS levels of cells were monitored using the DCFH-DA fluorescent probe assay. The MDA production and cellular activity ofLDH, CAT, T-SOD, and GSH-PX were measured by kits. The effect of WNWAD onthe protein levels of cell apoptosis hallmarks Bcl-2, cleaved caspase-3and PARP wasexamined using Western-blot analysis. The results showed that WNWAD possessedextraordinary oxygen radical absorption capacity (with an ORAC value=2.91±0.17μM TE/μM) in vitro. Then at cellular level, MTS results displayed that WNWADdose-dependently inhibited H2O2-induced cell oxidative stress in HEK293cells andfully recovered the oxidative damage induced by400μM H2O2at1.0μMconcentration.2′,7′-Dichlorofluorescin diacetate (DCFH-DA) fluorescent probe assayfurther proved that WNWAD inhibited cell oxidative stress by reducing intracellularROS accumulation in intact HEK293cells. In addition, scanning electron microscope(SEM) images showed that the morphology of HEK293cells under H2O2treatmentdisplayed a cell apoptosis phenotype, while WNWAD pretreatment prevented thisphenotype alternation. Furthermore, Western blot results indicated that WNWAD upregulated the anti-apoptotic protein Bcl-2level and down regulated the apoptosisexecutor protein cleaved caspase-3and cleaved PRAP level in H2O2-inducedoxidative stress HEK293cells.In conclusion, all above results suggested that pentapeptides from egg whiteprotected HEK293cells against H2O2-induced oxidative stress by inhibitingintracellular ROS accumulation and blocking ROS activated mitochondria-mediatedcell apoptosis pathway.