Protective Effect Of Whey Protein Hydrolysates On Hydrogen Peroxide-induced Oxidative

Natural antioxidative peptides have been paid more attention recently for its natural and safe properties, thus peptides from protein can be a good kind of potential antioxidant. Whey protein,as an excellent protein source, has been developed into a variety of bioactive peptide products. Recent studies have found whey protein possesses sequence of antioxidant activity; however, it was more concentrated on the hydrolysis optimization and primary purification process of antioxidant peptides from whey protein.In this study, whey protein was hydrolyzed by pepsin and trypsin,and treated by macrospores absorption resins. PC12 cells were seeded into culture plates for 24 h, and pretreated with whey protein hydrolysates (WPHs) at different concentrations for 2 h. Then Cells were washed and incubated with 100μM H₂O₂ in the presence of WPHs for another 24 h. The protective effects were studied by measuring the cell viability (by MTT assay), photographing the cell morphological alterations, detecting the activity of cell antioxidant enzymes and studying of apoptosis through mitochondria-mediated pathway. The result showed the relative molecular mass of WPHs was 369¹980 Da. Cell viability was measured by MTT assay, and result showed that 100⁴00μg/mL WPHs increased the percentage of viable cells by 20%³0% when incubated with H₂O₂ and kept a good state of cells without number reducing and cells congregating. The activity of cell antioxidant enzymes, which is detected by reagent box, was elevated in 200μg/mL WPHs group, that means WPHs could inhibit H₂O₂-induced antioxidant enzymes activity-decrease and kept the total antioxidation capacity. Cell apoptosis by mitochondria-mediated pathway was assessed and results showed that the percentage of apoptotic cells was reduced by 8.3%-19%, mitochondrial membrane potential was stabilized, and the elevation of intracellular calcium was suppressed. The activity of Caspase-3, determined by Caspase-3-regent box, was inhibited in 100μg/mL and 200μg/mL WPHs groups. Furthermore, the levels of apoptotic-related proteins, poly (ADP-ribose) polymerase (PARP), Bcl-2 and Bax were measured by western blot analysis, and the WPHs was found to block the H₂O₂-mediate degradation of Bcl-2 and the cleavage of PARP, decrease the expression of Bax. These results indicate that WPHs has potential protective effects against H₂O₂-induced oxidative stress in PC12 cells, which might be an agent for prevention and treatment of neurodegenerative diseases implicated with oxidative stress.The results of our study show that WPHs are endowed with significant protective effects against H₂O₂-induced oxidative stress in PC12 cells by their antioxidant ability, and might be a good antioxidant addition agent.