Research On The Role Of NF-?B In The Antioxidant Protective Effect Of Rice Peptide In Vitro

By-products which were generated in the production of cereal were overlooked and even wasted easily.But its protein content is rich,the composition of amino acid is reasonable.The small peptides,obtained by the degradation of the by-products with biological enzymes,have the biological activity of lowering blood pressure and lipid levels,enhancing immunity,anti-cancer and so on.Therefore,Rational utilization of small peptides in cereal has improved the value of its by-products.In this study,the effect and mechanism of oxidative stress sensitive factor-NF-KB on the antioxidant protection of rice peptide in vitro were studied.MTS method was used to determine the effect of rice bioactive peptides on the cell viability of HUVEC cells and the growth curve of cells in 24 h under the intervention the pyrrolidine dithio carbamate(PDTC)and H2O2.The effects on the morphological changes of HUVEC cells after the intervention of PDTC and H2O2 were observed by optical microscope,fluorescence microscope and electron microscope.The apoptosis of cells was detected by flow cytometry and Tunel kit.The changes of cell migration ability were observed and analyzed by the angiogenesis experiment;changes in the expression of the sensitive factor NF-?B before and after treatment was detected by Western blot,thus exploring the role of sensitive factor NF-?B in the antioxidant protection of rice bioactive peptides in vitro.The concentration of H2O2 and PDTC treatment of 24 h after 50%MTS treatment was 0.05 mmol/L and 0.06 mmol/L,respectively.On the basis of oxidative damage model in vitro,the concentration of H2O2 and PDTC which can make the oxidative damage rate of cell up to 50%(IC50)was 0.05 mmol/L and 0.06mmol/L respectively,selected by MTS method.Combined with previous studies and results,the best protective concentration of 0.2 mg/mL rice bioactive peptides was selected.Results of opticial microscopy showed that:the cells in normal control group(control group)and rice bioactive peptides group(RBP)group had regular cell shape,vigorous growth,but cells in injury group showed shrinkage in cell morphology,smaller size,and nucleolar spillover;after the protection of rice bioactive peptides,cells had similar morphology with normal control group,only a small part of cells showed the same cell morphology with injury group.Results of scaning electron microscopy showed that cells in the control group and RBP groups had rich surrounding microvilli,structural integrity,clear clearance,plasmodesmata connections;in injury group,the surface microvilli of cells were disappeared,nuclear condensation,the morphology of cell were changed,apoptotic body were formed.;protection group of cell surface microvilli,cell structure and the control group were similar.The surface microvilli of cells in protective groups were reappeared and the structure were similar with that of control group.Results of cell apoptosis detection showed that the cells in normal control group and rice peptide group grow vigorously,most of the cells in the injured group were in the state of apoptosis;after the addition of rice bioactive peptides,the apoptosis is significantly reduced,most of the performance is normal.Angiogenesis experiments obtained that;hollow microtubule bundles could be formed in the control group and RBP group,whereas the cells in the injured group were single and sprouting and intussusception did not appear;cells in the rice bioactive peptides protective group is partly in the state of dispersion,but most of them were clustered into microtubule bundles.Results of Western blot analysis showed H2O2 could up-regulate the expression of NF-?B/p65 protien while PDTC down-regulated NF-?B expression levels.The expression of NF-?B was down-regulated in rice bioactive peptides-H2O2 treatment group,and inhibited cells apoptosis through the inhibition of NF-?B nuclear translocation,thus playing a protective role.The expression of protein was obviously up-regulated in rice bioactive peptides-PDTC treatment group.After phosphorylation modification,NF-?B was activated to carry out nuclear transfer and then regulated cell apoptosis.NF-?B plays an important regulatory role in the oxidative stress reaction.The results show that When HUVEC was damaged by the external environment stimuli,the rice bioactive peptides can help protect against oxidative stress.The possible mechanisn is When cells are encountered in different types of oxidative damage,rice bioactive peptides can regulate the nuclear translocation of NF-?B,and affect the expression of NF-KB-p65,and then inhibit the oxidative damage of the cells.These results provide an important theoretical basis for the development of rice bioactive peptides to be antioxidant functional food or as a functional factor additives and the development of therapeutic agents for the treatment of cardiovascular diseases,and also provide the basis for the development and utilization of the by-products of the deep processing of rice.