| Oxidative stress is the major cause of aging and neurodegenerative diseases. Under normal conditions, there is a integrated set of antioxidant system in the human body. However, when oxygen free radicals 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, antioxidant were prepared and filtered, which had been isolated and identified from wheat germ in recent years. The results showed that antioxidant peptide of wheat germ RVF took on the best protective effect in the oxidative stress-induced cells damage, and it was chosed for further study. To better understand the wheat germ peptide RVF antioxidant mechanism, oxidative damage model from SH-SY5Y cells was established by H2O2. In order to survey RVF on H2O2-induced cytotoxicity in SH-SY5Y, we investigated cell viability, lactate dehydrogenase (LDH) release rate and cell morphology, the results showed that the RVF pre-incubated cells survival rate was increased by 15.91%, LDH release rate reduced by 20.00% and cell morphology had been significantly improved.The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were examined by colorimetric methods, the results showed that antioxidant peptide of wheat germ RVF pretreated elevated total antioxidant capacity by 27.33% and also significantly reduced the content of MDA. ROS generation, mitochondrial membrane potential and Ca2+ influx were detected by flow cytometry, results showed that the the RVF pretreatment could effectively inhibit the elevation of ROS, maintain normal mitochondrial membrane potential and calcium homeostasis. Apoptosis was assessed by nuclear staining with fluorescent DNA binding dye, Hoechst 33342 and PL results showed RVF pretreatment mitigated these morphological changes, inhibited the formation of apoptotic bodies. Apoptosis was also quantitatively analyzed by flow cytometry, RVF treatment significantly reduced the proportion of apoptotic cells induced by H2O2 and the apoptotic rate decreased by 10.81%. SH-SY5Y cells apoptotic protein were tested by western blot including Bcl-2, Bax, PARP and caspase-3, RVF pre-incubation promoted expression of Bcl-2 and inhibited expression of Bax, but Bcl-2/Bax ratio changed little at a low concentration, and elevated 18.67% and 24.97% in high concentrations. Furthermore, RVF inhibited H2O2-induced expression of the active form of caspase-3, which resulted in the decreased expression of the cleaved-RARP. In a word, antioxidant peptide of wheat germ RVF inhibited neuronal apoptosis induced by H2O2.In conclusion, the results of our study indicated that antioxidant peptides of wheat germ could significantly protect SH-SY5Y cells against H2O2-induced oxidative damage. The specific protective mechanisms including improving the intracellular total antioxidant capacity, inhibiting lipid peroxidation and accumulation of intracellular ROS, maintaining normal mitochondrial membrane potential and calcium homeostasis, increasing the expression of anti-apoptotic proteins, as well as preventing the mitochondrial pathway. Accordingly, our results showed that antioxidant peptides of wheat germ could be developed as potential anti-oxidant for food or pharmaceutical fields. |
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