| Seven active peptides with definite amino acid sequence isolated from hazelnut protein hydrolysis were tested for their protecting effect on oxidative stress injury induced by Ang?solution in human umbilical vein endothelial cells?HUVECs?through in vitro chemistry and cell experiments.Firstly,the antioxidant activities of 7 hazelnut active peptides?Vc as control?were investigated by detecting the scavenging on DPPH and ABTS+·free radicals.The results showed that the 7 active peptides all showed a peptide concentration dependent relationship on the DPPH and ABTS+·radicals scavenging activities.Antioxidant capacity increased as the increased peptide concentration.Glu-Trp?EW?peptide showed high DPPH and ABTS+·free radicals scavenging rates with values of?91.52±3.35?%and?92.48±1.69?%,respectively,at the concentration of 1mg/mL.Furthermore,its antioxidant activity was the highest among 7peptides,and was closed to Vc.Another 6 peptides including Asp-Trp-Asp-Pro-Lys?DWDPK?,Ala-Trp-Asp-Pro-Glu?AWDPE?,Ala-Asp-Gly-Phe?ADGF?,Ser-Gly-Ala-Phe?SGAF?,Glu-Thr-Thr-Leu?ETTL?,and Ala-Gly-Gly-Phe?AGGF?showed DPPH and ABTS+·free radicals scavenging rates of?68.25±1.25?%,?63.38±2.43?%,?59.57±1.47?%,?49.63±0.61?%,?43.96±1.31?%,?39.22±3.18?%and?78.41±2.95?%,?71.38±2.50?%,?69.38±1.96?%,?56.89±3.31?%,?52.17±2.61?%,?46.05±1.48?%,respectively,at the concentration of 1mg/mL.Seven peptides have different antioxidant capacities may due to their different amino acids composition.For example,the peptides containing Asp,Trp and Phe amino acids are strong for DPPH free radical scavenging.Pro?hydrophobic acid?can significantly improve the antioxidant activity of peptides.Trp?hydrophobic acid?has the greatest capacity for ABTS+·free radical scavenging.Secondly,the effects of 7 peptides on growth and reproduction of oxidative stress injury HUVECs induced by Ang?were investigated by MTT assay.The results showed that different concentrations of 7 peptides solution had neither inhibitory nor promoting effects on the proliferation of HUVECs;The optimal injury concentration of Ang?solution for establishing oxidatively damaged HUVECs model was 10-7 mol/L,and the optimal induced time was 6h.Under this condition the HUVECs viability reduced to approximately 0.534 times of the normal growth cells.HUVECs were significantly increased after treated by 7 peptides solution for 18h,following by treated with final concentration of 10-7 mol/L Ang?solution for 6 h.Overall,the 7peptides solution have protective effect against Ang?induced oxidative stress injury HUVECs.Based on the in vitro results,EW,ADGF,AWDPE and DWDPK were selected for the further research on protective mechanism in oxidative stress injury HUVECs by morphologic observation and antioxidant effects.The intracellular ROS was monitored by using the DCFH-DA fluorescent probe labeling assay labeling and used BD FACSCalibur flow cytometer detection of FITC-A subset.Result:4 peptides inhibited the morphology changes of HUVECs induced by Ang?solution,and they could down-regulate the production of LDH and MDA and up-regulate the activity of intracellular CAT,SOD and GSH-PX levels compared to Ang?solution group.The 4 peptides could scavenge over-generated ROS in HUVECs to protected HUVECs from oxidative stress injury.Finally,real time quantitative reverse transcriptive polymerase chain reaction?qRT-PCR?was used to determine the expressions of NOX4 and p22phox mRNA.Western blot was used to evaluate the protein expression of NOX4 and p22phox.The results showed that 4 peptides including EW,ADGF,AWDPE and DWDPK could protect HUVECs under the oxidative stress induced by Ang?solution,reduceNOX4 and p22phox levels,control the activity of NADPH oxidase,and inhibit ROS generation in HUVECs.Overall,these 4 peptides indicated regulated NADPH activity and ROS production by reducing NOX4 and p22phox levels in HUVECs. |
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