The Inhibitive Effect Of Peptides From Rice ?-globulin On Vascular Cell Dysfunction And Mechanism Research

In recent years,the effects of rice protein in preventing chronic diseases have been confirmed,such as anti-diabetes,anti-hypertension and anti-atherosclerosis(AS).Globulin is the most important bio-active protein of rice,and the majority of rice globulin is ?-globulin.The anti-atherosclerotic role of rice ?-globulin(100 mg/day/kg body weight)has been proved in our previous study,but the key fragments of peptides after gastrointestinal digestion and absorption remains unclear.Although it has been confirmed that the prevention of ?-globulin did not performed by regulating lipid metabolism,its exact mechanism still need to be further studied,to clarify whether ?-globulin performed by protecting vascular cells,as another most important anti-atherosclerotic mechanisms except the way mentioned above.Therefore,this paper studied the preparation of the rice ?-globin peptide and its protective effect on vascular cells in order to reveal the anti-AS mechanism of the rice ?-globulin.First,the absorbed part of the digestion product of rice ?-globulin in vitro was separated and purified using Sephadex G-15 gel chromatography and Semi-preparative HPLC in sequence,and 11 main components were collected respectively.Then the amino acid sequence was determined by LC-ESI-MS,and the secondary mass spectrograms showed that 5 of them(YYGGEGSSSEQG,GEQQQQPGM,YGEGSSEEG,AAGALPS and SESEM)were matched with Uniprot database.The target peptides of purity 97% were synthesized by solid phase synthesis.In order to verify the absorbability of the target peptides,10 mg/kg weight/day dose gavages of the target peptides were given to the golden hamsters,and 4 hours later the plasm was collected for investigating the presence or absence of the target peptides in the serum.The results of MS showed that all the five target peptides could be detected in the serum respectively and it proved that all target peptides can be absorbed directly by the body.Second,the effects of the 5 target peptides derived from rice ?-globulin on vascular endothelial cells injury were studied.The cells model was established by 10 ng/m L tumor necrosis factor-?(TNF-?)for 6 hours.The study was performed on Human Umbilical Vein Endothelial Cells(HUVEC)that were pretreated with 50 ?g/m L of peptide for 18 hours.The cells viability and indicators of Inflammation and oxidative stress were measured.As the results,the decreased viability of HUVECs induced by TNF-? was significantly increased by peptide.The peptide reduced the levels of inflammation factor,as nitric oxide(NO),inducible nitric oxide synthase(i NOS),intercellular adhesion molecule-1(ICAM-1),and vascular cell adhesion molecule-1(VCAM-1)obviously.It also affected antioxidant indexes effectively including decreasing the levels of malondialdehyde(MDA),and reactive oxygen species(ROS)while increasing the activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD).Furthermore,peptides inhibited nuclear translocation and expression of NF-?B p65 protein by decreasing inhibitor of ?B kinase ?(IKK?)protein and inhibiting degradation of I?B? protein.Apoptosis of HUVECs induced by TNF-? was significantly increased by YGEGSSEEG,which also regulated expression of Bcl-2/Bax proteins and inhibited phosphorylation of p38 protein.These results demonstrated that the peptides reduced TNF-?-induced vascular endothelial cells injury by inhibiting inflammatory response and oxidative stress,thereby reducing the risk of atherosclerosis.The results in migration of VSMC showed that YYGGEGSSSEQG and SESEM peptide could effectively inhibit the proliferation and migration of VSMC.The results of Western blot show that the peptides can down regulate the PDGF-BB,which could be the key to inhibit the proliferation and migration of VSMC.Third,to further verify the efficacy of rice ?-globulin in the prevention of AS based on vascular cell protection,YYGGEGSSSEQG and SESEM(10 mg/kg weight/day)were administered to Apo E-/-mice for three months.The results of aortic staining showed that both the two target peptides significantly decreased the atherosclerotic(AS)plaque area of aorta in Apo E-/-mice,and the serum test results illustrated the significantly decrease of GSH-Px,SOD,CAT,as well as VCAM-1,while without effects on serum and liver lipids.The ?-globulin peptides were separated and identified.The biosynthetic peptides were tested in vivo and in vitro,which confirmed their vascular cell protection include repairing the TNF-?-induced HUVEC injury and inhibiting VSMC proliferation and migration,and the anti-AS mechanism.