| In the production process of grain, many byproducts were generated which were overlooked even wasted,what’s more, the composition of amino acid of rice protein was reasonable. Using the method of biological, it can be degraded to peptides. Among of peptides have biological activities, such as lowering blood pressure, enhancing immune, anti-cancer. Therefore, it is a good way to use of byproducts effectively and fully, but also provides the theoretical basis for the application of active peptide as new health factors. In this study, the antioxidant of active peptide from rice residue to human umbilical vein endothelial cells was investigated and preliminary analysis of its mechanism with NF-kB in vitro. MTS method under the influence of PDTC and H2O2 intervention was used to determine rice peptides effect on HUVEC cells survival rate and growth curve. Using optical microscopy, fluorescence microscopy, electron microscopy analysis of the impact on the morphological changes of HUVEC after PDTC and H2O2 intervention; Flow cytometry assay and Tunel were used to investigate the cell apoptosis. The experiment of angiogenesis was used to measure the ability of cells migration. Western blot was used to detect the change of expression of NF-kB before or after treatment, in order to research and explore the antioxidant mechanism of rice peptides in NF-kB in vitro.Experimental results show that on the basis of oxidative damage model in vitro, MTS was used to filter out the concentration of IC50 with H2O2 and PDTC dealing 24 hour, the concentration of IC50 were 0.05 mmol/L,0.06 mmol/L,respectively. According to preliminary studies selecting the concentration of rice peptide of 0.2 mg/mL is the best protection concentration.Result of electron microscope showed that the cell of normal control group and rice peptides group were polygon,vigorous growth,uniform size,clear edge,abundant cytoplasm and had 2-6nuclears in each cell.A part of cell of induced groups were apoptosis,morphology shrinkage, the cell were smaller, nucleus became blurred even nucleoli were spilled. Pretreatmen with rice peptides, the cell is similar to control group,only a small portion of cell as induced groups. Result of scaning electron microscopy showed that there were rich in microvilli on the surface of normal control group and rice peptiede group,the cell intervals and structure were clear,the gap junction intercellular rich.The surface microvilli of induced groups were disappeared, nuclear condensation, the shape of cell changed, the apoptosis were formed. The surface microvilli of protective groups were occurred again and the structure as control cell.Detection of apoptosis derived that the cell growth of normal control group and rice peptide group were vigorous, the majority of cell were apoptosis in induced groups while the phenomenon of apoptosis were significantly reduced and most as control group in the protective groups. Angiogenesis experiment can be seen that There were a hollow microtubule bundles in the normal control group and rice peptides group. The induced groups were single and the phenomenon of sprouting or intussusception didn’t show. Although a part of cell of protective groups were in dispersed state, most had the ability of formation microtubule bundles.Western blot analysis showed H2O2 can up-regulate the expression of NF-icB/p65 protien while PDTC can down-regulate H2O2-induced inhibit the levels within overall range.With rice peptides protection H2O2-induced inhibit the nuclear translocation of NF-kB and the cell apoptosis. The group of rice peptides protected PDTC significantly increased which is opposite with the group of rice peptides protected H2O2, so we can speculate that its mechanism is not the same, but can achieve the same or similar protective effect. The protein expression of phos-NF-KB was significantly decreased in each groups, which may be activated the ability of transferring nuclear to regulate apoptosisNF-kB plays an important regulatory role in the oxidative stress process, the results show that NF-kB is quite important in the antioxidant of rice peptides in vitro. When the external environmental stimuli and damage HUVEC, rice peptides inhibit cellular oxidative stress and protect from damage, The possible mechanisn is when different types of oxidative damage were coming, rice peptides inhibit the nuclear translocation of NF-kB,thereby the expression of NF-KB-p65 gene was suppressed to inhibit cellular oxidative damage.This will be developed rice peptides into rice antioxidant function food or as a functional factor for cardiovascular disease.lt is not only provided important theoretical basis to develop therapeutic drugs,but also lay a foundation of utilization and development of deep processing of rice byproducts. |
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