Protective Effects And Its Mechanism Of Catalpol On The H₂O₂-induced Oxidative Damage In INS-1 Cells

[Objectives]At first,molecular docking predicted the effective components and protein targets of Gegen Qinlian Decoction（GQD）intervened in the oxidative stress of type 2 diabetes（T2DM）,and simulates and verified the binding activity of these effective components and protein targets.Thus in vitro experiments was performed to verify the antioxidant mechanism of catalpol,one of important anti-oxidative component,on the INS-1 cell with the H₂O₂induced oxidative damage,which could improveβ-cell function.It would explore the action mechanism of catalpol in treating T2DM.[Methods]1.The effective components and proteins of GQD involving to the oxidative stress of type2 diabetes were obtained by molecular docking.2.The INS-1 cells with the H₂O₂induced oxidative damage model:Different concentrations of H₂O₂(25,50,100,200,400μmol·L^-1)were used to induce INS-1 cells at different times（0.5h,1h,2h,4h）to establish the H₂O₂induced INS-1 oxidative damage model.The CCK-8 method was used to detect cell viability of INS-1 cells.3.According to the prediction result of molecular docking,catalpol was chosen as the experimental drug.The CCK-8 method was used to detect the effect of catalpol with different concentrations(1,5,10,20,40,80,160μmol·L^-1)on the cell viability for 24 hours pre-treatment.According to the above results,and then selected the range of catalpol concentration(1-80μmol·L^-1)for 24h pre-treatment on the H₂O₂ oxidative damage model,cell viability at different concentration of catalpol(1,5,10μmol·L^-1)by CCK-8 method.4.Effects of catalpol on the antioxidant enzymes and relative metabolites of INS-1 cells including:normal group,model group,positive group(NAC,500μmol·L^-1),and 1,5,10μmol·L^-1 catalpol groups.After 24hours pre-treatment with different concentrations of catalpol and NAC,the content of reactive oxygen species（ROS）were detected in INS-1 cells by DCFH-DA fluorescent probe;whereas cell antioxidant capacity and lipid peroxidation level were respectively detected by superoxide dismutase（SOD）kit and malondialdehyde（MDA）kit.5.The effect of catalpol on the apoptotic effect of INS-1 cells with the H₂O₂induced oxidative damaged model:AO-EB double staining was used to observe the effect of catalpol on the morphology of INS-1 cells;Annexin V-FITC/PI staining and flow cytometry were used to detect the anti-apoptotic effect of catalpol on the oxidatively damaged INS-1 cells.6.The catalpol of oxidative damage to INS-1 cells causes changes in the expression of antioxidant proteins（p-Nrf2（Ser40）/Nrf2、Keap1、HO-1、p-ERK1/2（Thr202/Tyr204）/ERK1/2）,insulin secretion and related proteins（PDX-1、GIUT2）on the INS-1 cells with H₂O₂-induced oxidative damage model by western blot.ELISA detects the insulin secretion content of each group under the conditions of 3mmol/L basal glucose（BIS）and 30 mmol/L high glucose culture medium（GSIS）.[Results]1.The molecular docking analyzes the interaction between these active ingredient targets of four herbs from GQD and the oxidative stress targets of T2DM.Combined with literature researches,6 effective antioxidant components of GQD including catalpol,quercetin,apigenin,kaempferol,baicalein and baicalein were verified by molecular docking simulation,including oxidative targets such as Keap1（5NLB）,ERK1/2（1M3G）,and PDX-1（2HIK）protein.The binding activity of catalpol with the key antioxidant protein Nrf2 scores was 8.368,which indicated that catalpol had a strong binding activity with Nrf2 as an antioxidant.2.Cell viability experiments:INS-1 cells were induced by different concentrations of H₂O₂(25,50,100,200,400μmol·L^-1)with different time（0.5h,1h,2h,4h）.Compared with the normal group,the survival rates of INS-1 cells were 92%（P<0.05）,74%（P<0.001）,50%（P<0.001）,49%（P<0.001）,48%（P<0.001）after 2h treatment with 25,50,100,200,400μmol·L^-1 H₂O₂.Finally,50μmol·L^-1 H₂O₂ for 2h intervention was considered as the appropriate inducing concentration and time.3.1μmol·L^-1 to 80μmol·L^-1catalpol for 24 hours pretreatment with from had no significant effect on cell viability.Compared with the model group,low concentrations(1,5,10μmol·L^-1)had anti-oxidant protection effects on the H₂O₂induced oxidative damage model（P<0.001）.4.The results the relative content of ROS in the normal group,positive group and catalpol groups(1,5,10μmol·L^-1)after 24hour pretreatment was（1±0.15,2.71±0.16,1.78±0.21,2.43±0.10,1.96±0.27,1.82±0.34）by the DCFH-DA fluorescent probe experiment.Compared with the model group,all other groups had statistical difference（P<0.001）.The intracellular SOD content(U·mg^-1)in model group,normal group,positive group,and catalpol groups were 274.19±10.99,232.78±5.46,265.01±11.37,238.46±6.82,249.70±10.85,265.25±13.15 respectively.Compared with the model group,normal group,positive group,10μmol·L^-1catalpol had statistical differences（P<0.001）.In addition,the intracellular MDA content(nmol·mg^-1)in the model group,normal group,positive group,and catalpol groups(1,5,and 10μmol·L^-1)were（0.80±0.10,2.16±0.31,1.06±0.08,1.48±0.21,1.63±0.17,1.24±0.23）respectively.Compared with the model group,normal group（P<0.001）,positive group（P<0.001）,catalpol group including 1μmol·L^-1（P<0.001）,5μmol·L^-1（P<0.01）,10μmol·L^-1（P<0.001）were statistically different.5.The anti-apoptosis effect of catalpol on the INS-1 cells with H₂O₂induced oxidative damage model:（1）AO-EB double staining morphology experiment results:most of cell morphology is normal with occasional apoptotic cells in the normal group whereas Compared with the model group,the apoptosis and oxidative damage of catalpol groups(1,5,10μmol·L^-1)and positive group were gradually reduced.（2）Flow cytometry detection of apoptosis experiment:The intracellular apoptosis rate（%）of the model group,normal group,positive group and catalpol groups(1,5,10μmol·L^-1)were8.02±1.94,51.6±1.32,13.07±0.21,36.13±2.28,24.7±0.81,18.23±0.75 by Annexin V-FITC/PI staining method.Compared with the model group,normal group,positive group and(1,5,10μmol·L^-1)catalpol were statistically different（P<0.001）.6.The antioxidant protein and insulin secretion-related protein expression levels:Compared with the model group,the expression of p-Nrf2（Ser40）/Nrf2 in the normal group,positive group and catalpol groups(1,5,10μmol·L^-1)were increased significantly,which have statistical difference.Compared with the model group,the protein expression levels of Keap1 in the normal group and catalpol groups(1,5,10μmol·L^-1)were increased significantly,which have statistical difference.Compared with the model group,the protein expression levels of HO-1 in the normal group,positive group and catalpol groups(1,5,10μmol·L^-1)was increased significantly,which have statistical difference.Compared with the model group,the normal group,the protein expression of levels p-ERK1/2（Thr202/Tyr204）/ERK1/2 in the normal group,positive group,catalpol groups(1,5,10μmol·L^-1)were increased significantly,which have statistical difference.Compared with the model group,the protein expression of levels of PDX-1 in the normal group,positive group and(5,10μmol·L^-1)catalpol groups were increased significantly,which have statistical difference.Compared with the model group,the protein expression of levels of GLUT2 in the normal group,positive group and(1,5μmol·L^-1)catalpol groups were increased significantly,which have statistical difference.In the BIS/GSIS state,compared with the model group,the normal group,the positive group,and the catalpol groups(1,5,10μmol·L^-1)insulin secretion increased.[Conclusions]1.50μmol·L^-1 H₂O₂ with 2 hours treatment was used to establish an oxidative damage model of INS-1 cells.2.Catalpol improved cell viability and reduced apoptosis cell of H₂O₂-induced oxidative stress damage model and reduces of INS-1 cells.3 Catalpol effectively regulated the activities of SOD and MDA.In addition,catalpol activated the ERK/Keap1-Nrf2/HO-1 antioxidant pathway and increased the proteins expression levels of PDX-1 and GLUT2.These results suggested that catalpol could improve cell damage caused by oxidative stress to restore INS-1 cell insulin secretion function.