| Oxidative stress is one of the major trigger of aging and neurodegenerative diseases. Under normal conditions, there is a integrated set of antioxidant system in the human body and oxygen free radicals (ROS) will not be invasive. However, when ROS are generated too much or metabolic disorders cause disfunction of the endogenous antioxidant defense system, the excess oxygen free radicals involved in the process of oxidation of biological macromolecules will cause the body’s oxidative damage, finally result in oxidative injury. Nevertheless, if antioxidants are supplied to scaveng free radical, oxidative stress can be inhibited. Now wheat germ peptides had been reported to own antioxidant capacity in vitro and in vivo. Though this was researched in vitro or in traditional animal experiments, the study on the antioxidant protection mechanism of oxidative damage to cells has not been reported yet. In the present study, H2O2-induced oxidative stress model in human neuroblastoma cell line SH-SY5Y cells is used to investigate the neuroprotective effects and its potential mechanisms of antioxidan peptide of wheat germ.Firstly, RVF, peptide of wheat germ isolated and identified earlier, was chosen as the antioxidant used in the research because of its better capacity of antioxidation. To better understand the antioxidative mechanism of wheat germ peptide RVF, research used SH-SY5Y cells treated with H2O2 as the oxidative injury model cells. In order to explore the effect of RVF on H2O2-induced cytotoxicity in SH-SY5Y, we investigated cell viability and cell morphology, and the results showed that pre-incubation with the 200 μmol/L RVF for 12 h could increase cell survival rate by 21.97% and improve morphology of the cell significantly. The study explored ROS level by flow cytometry and Malonaldehyde(MDA) was measured by Colorimetry. The MDA content were determined by colorimetric methods, and the results showed that antioxidant peptide of wheat germ RVF pretreated significantly reduced the content of MDA. Interestingly,200 μmol/L RVF could decrease content of MDA from 2.74±8.63%(p<0.01) to 1.35±16.60%(p<0.01). Intracellular oxygen free radicals was detected by flow cytometry. As the result showed, the ROS level of group treated with H2O2 had a significant increase by 9.97 ± 12.13%(p<0.01) compared with the control group. The rate of ROS level of group treated with 100,150,200 μmol/L wheat germ antioxidant peptide RVF, compared with control group, decreased to 5.72 ± 17.83%(p<0.01),4.21 ± 18.29 (p<0.01), 2.54 ± 37.00%(p<0.01) respectively.The study explored the mitochondrial membrane (MMP) by flow cytometry. RVF could successfully maintain the MMP, reduce the MMP negtive cells from 92.58%±9.4%(p<0.01) to 85.98%±8.6%(p<0.01),67.13%±7.4%(p<0.01),44.58%±5.32%(p＜0.01).Apoptosis was also assessed by detecting apoptotic protein through western blot method. RVF could efficiently decrease the activation of MAPK pathway through phosphorylation of JNK and p38. The detected protein included Bcl-XL, Bax,cytochrome c, PARP and caspase-3, phosphorylation of protein(JNK, p44/42,p38)in MAPK signal pathway, it was revealed that RVF pre-incubation recovered the expression of Bcl-XL and inhibited translocation of Bax on mitochondria and decreased the release of the cytochrome c. Furthermore, RVF inhibited H2O2-induced cleavage of caspase-3 known as the active form, which resulted in the decreased expression of the cleaved-RARP. Besides, phosphorylation of JNK, p44/42 and p38 was also remarkably decreased with the existence of RVF. In a word, antioxidant peptide of wheat germ RVF inhibited the neuronal apoptosis induced by H2O2.In conclusion, the results of our research indicated that antioxidant peptides of wheat germ could significantly protect SH-SY5Y cells against H2O2-induced oxidative injury. The specific protective mechanisms including inhibiting biomacromolecule oxidation and accumulating intracellular ROS, maintaining normal mitochondrial membrane potential, increasing the expression of anti-apoptotic proteins, as well as preventing the activation of the mitochondrial apoptosis pathway. Accordingly, we supposed that antioxidant peptides of wheat germ could be developed as potential anti-oxidative product in food or pharmaceutical fields. |
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