Protective Effects And Its Mechanism Of Fish Roe Polypeptide On H₂O₂-induced Oxidative Damage In INS-1 Cells

Objective:To explore the hypoglycemic activity of fish roe polypeptide and its protective effect on oxidative damage of INS-1 cells and its molecular mechanism,provide experimental data and theoretical support for its development and utilization.Methods:1.The effect of fish roe polypeptide on H₂O₂-induced oxidative damage in INS-1cells?1?Blank control group,model group,fish roe polypeptide?25,50,75,100,150,200?g/mL?and positive control?quercetin 6,25?g/mL?were set in this study.The cell viability and the IC₅₀ were detemined by MTT assay.?2?Then the oxidative damage cell model was established by H₂O₂ and the protective effect of fish roe polypeptide on the oxidative damage of INS-1 cell was detected by MTT assay.The insulin secretion cell model was established by treating the cell with 5.6 and 16.7mmol/L of glucose and the effect of fish roe polypeptide on the insulin secretion of INS-1 cells in oxidative damage was determined by ELISA.Futhermore,the effects of fish roe polypeptide on ROS and cell morphology of oxidative damage INS-1 cells were detected by DCFH-DA fluorescent probe and Hochest 33258 staining respectively.2.Molecular mechanism of protective effect of fish roe polypeptide on H₂O₂-induced oxidative damage in INS-1 cells?1?Blank control group,model group,fish roe polypeptide?25,50,75,100,150,200?g/mL?and positive control?quercetin 6,25?g/mL?were set in this study.The content of SOD and GSH in INS-1 cells was detected by micro-method method,and the content of CAT and GSH-Px in cells was detected by u-ltraviolet spectrophotometry.?2?Blank control group,model group,fish roe polypeptide?50,100,150?g/mL?and positive control?quercetin 6?g/mL?were set in this study.The erpression of HO-1,GCLC,NQO1,UCP2,ERK1/2 and nuclear expression of Nrf2of INS-1 cells in oxidative damage by using ERK1/2 blocker U0126 were analyzed by western blot assay.Cell viability was detem-ined by MTT assay and insulin secretion was detemined by ELISA for INS-1 cells in oxidative damage by using ERK1/2 blocker U0126.Results:1.The effect of fish roe polypeptide on H₂O₂-induced oxidative damage in INS-1cells?1?Compared with the blank control group,the fish roe polypeptide had a proliferative effect on normal INS-1 cells.The 150?g/mL treatment group had the best effect,and the cell viability was?119.41±3.59?%,which was significantly higher than that of the blank control group?100.00±1.26?%?P<0.05?.?2?H₂O₂ inhibited cell proliferation with IC₅₀0 value of 840?mol/L;fish roe polypeptide had a protective effect on INS-1 cell viability oxidative damage,150?g/mL treated group was the highest cell viability?89.74±3.70?%was significantly higher than the model group?50.40±4.33?%;when the concentration of fish roe polypeptide was 200?g/mL,the BIS of oxidatively damaged INS-1cells was?81.45±1.45?%significantly higher than that of the model group?63.83±1.39?%?P<0.05?.The fish roe polypeptide 100?g/mL treatment group had the best protective effect on oxidative damage of INS-1 cells GSIS,and its GSIS was?109.78±2.26?%significantly higher than the model group?63.83±1.39?%,the difference was statistically significant?P<0.05?.The results of ROS showed that the fish roe polypeptide had a scavenging effect on ROS in oxidatively damaged INS-1 cells.The ROS scavenging activity was the strongest at a concentration of 150?g/mL and the relative fluorescence intensity was?294.00±16.37?significantly lower than that of the model group?982.60±47.01??P<0.05?.Nuclear staining results indicate that fish roe polypeptides have protective effects on cell morphology integrity.Therefore,the fish roe polypeptide has the protective effect on oxidative damage of INS-1 cells.2.Molecular mechanism of protective effect of fish roe polypeptide on H₂O₂-induced oxidative damage in INS-1 cells?1?The content of SOD,CAT,GSH and GSH-Px in INS-1 cells was significantly higher than that in the model group after different concentrations of fish roe polypeptide?P<0.05?.?2?After H₂O₂ oxidative damage,the expression of Nrf2 in the model group and the expression of downstream protein were significantly down-regulated,and the difference was statistically significant?P<0.05?.After the intervention of fish roe polypeptide,the expression of Nrf2 in the nucleus of oxidatively damaged INS-1 cells was up-regulated in the 150?g/mL group,and the expression levels of HO-1,GCLC,NQO1 in the downstream of Nrf2 signaling pathway were up-regulated and the expression level of UCP2 and ERK1/2 protein in the treatment group of fish roe polypeptide was significantly up-regulated in a dose-dependent manner;after U0126 processing,the nuclear expression level of Nrf2decreased,and the fish seed peptide had protective effect on the cell viability and insulin secretion of oxidized INS-1 cells.The difference was statistically significant?P<0.05?,but its protective effect was lower than U0126.Unprocessed group.Conclusion:The fish roe polypeptide has a protective effect on oxidative damage to INS-1 islet cells,which protects INS-1 cells from H₂O₂-induced oxidative damage through the Nrf2-ARE pathway,and ERK1/2 mediates the protective effect of fish roe polypeptide on nuclear expression of Nrf2,cell viability and insulin secretion of oxidatively injured INS-1 cells.