| Oxidative stress plays an important role in neuronal cell degeneration observed in neurodegenerative diseases, which can lead to the damage of biomacromolecule, metabolism disorders and neurotransmitter transmission. Therefore, current attention has been dedicated to find natural neuroprotective agents that can reduce oxidative stress in neurons. Soybean protein as the starting point, aimed to evaluate the potential neuroprotective effect of food-derived peptides. The research was granted by Twelfth-Five National Science and Technology Support Program(NO.2012BAD33B03). Soybean protein as raw material, through hydrolysis, dialysis and membrane separation purification. PC12 cells exposed to H2O2 as a model to select the optimal components by cell viability and Plasma Membrane Damage. Evaluation of the antioxidant capacity of soybean peptides, measured a variety of cell specific marker to analysis the neuroprotective pathway of soybean peptides.(1) Preparation of soybean peptides is the basic in this study, soybean protein isolate was hydrolyzed with Alcalase, hydrolysates were sequentially separated by membrane ultrafiltration into four molecular size fractions, SPHs1(> 30 k Da), SPHs2(10~30 k Da), SPHs3(3~10 k Da) and SPHs4(0~3 k Da). It was necessary to evaluate the cytotoxic effect or proliferation effect of the hydrolysate fractions on PC12 cells before further assays were carried out. It was indicated that the fractions did not show cytotoxicity or stimulation of the proliferation of PC12 cells over 24 h as measured by the MTS assay. Then H2O2-induced injury on PC12 cells was assessed. The H2O2(25~800 μmol L-1) treatment decreased the cell viability in a dose-dependent manner with 42.91±4.39 % viability at 200 μmol L-1 H2O2 challenge which was used for further experiments.(2) The neuroprotective effects of the hydrolysate fractions were investigated. The cells pretreated with various SPHs(0.1~1.0 mg/m L) for 24 h before 200 μM H2O2 treatment(12 h) shown that the cell toxicity was markedly decreased. The highest effect was observed in pre-incubations with SPHs4(p<0.01) in a dose-dependent manner. Meanwhile, SPHs4 significantly decreased LDH release to 26.54 ± 1.85% at 1.0 mg/m L with H2O2-induced group(p<0.05). After the addition of SPHs4 into the cell culture with H2O2 treatment, cells grew better, the number of adhered cells increased, the number of round cells decreased, and cell synapse recovered normally. These data shows that SPHs4 could offer the protection against H2O2-induced cell death, and SPHs4 was selected for the further investigations.(3) We used three antioxidant methods which were closely related to neuroprotection to estimate the antioxidant activities of soybean peptides, including Fe2+ chelating, ORAC and inhibition of linoleic acid autoxidation assays. The SPHs4 had the strongest antioxidant activity. The SPHs4 provided the highest ORAC value of 235.1 μmol TE/g which was distinctly higher than the other fractions. The SPHs4 provided the highest Fe2+ chelating of 7.37 mg EDTA/g. SPIHs4 showed the highest antioxidant activity, with about 76.31 % inhibition of linoleic acid peroxidation. Neuroprotection and antioxidant capacity were related, which is important for the analysis and screening of natural neuroprotective agents.(4) PC12 cells exposed to H2O2 as a model to measure a variety of cell specific marker to analysis the neuroprotective pathway of soybean peptides. The results indicated that pretreatment of PC12 cells with SPIHs4, prior to H2O2 exposure, significantly increased the activities of superoxide dismutase(SOD) and catalase(CAT), and reduced the levels of MDA. Intracellular accumulation of ROS were also inhibited by SPHs4 treatment. These findings suggest that the protective effects of SPHs can be attributed to some peptides’ ability to neutralize radicals and other reactive oxygen species thereby enhance certain antioxidant enzymes while minimize the accumulation of toxic compounds. |
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