Protective Effect Of Metformin Combined With Dapagliflozin On Sirolimus-Induced Pancreatic Cell Injury

Objective: To investigate the protective effect of metformin(MET)combined with dapagliflozin(DAPA)on sirolimus(SRL)-induced pancreatic cell injury and its mechanism.Methods: 1.Taking INS-1 cells as research subjects,normal control(VH)group,SRL group,SRL+DAPA group,SRL+MET group and SRL+MET+DAPA group were assigned;2.The activity of INS-1 cells treated with different concentrations of drugs was detected by CCK-8 method,and the appropriate concentration of drugs was screened out;3.The activity of INS-1 cells in VH group and each experimental group was detected by CCK-8 assay;4.Glucose-stimulated insulin secretion assay(GSIS)was used to determine the insulin secretion level of INS-1 cells in VH group and each experimental group;5.Fluorescence staining was used to detect the reacive oxygen species(ROS)expression of INS-1 cells in VH group and each experimental group;6.Annexin V-FITC/PI kit was used to detect cell apoptosis in VH group and all experimental groups;7.The protein expressions of Mn SOD,Casepase-1,AMPK,PI3 K,AKT and Fox O-1 in INS-1 cells of VH group and each experimental group were detected by western blotting.Results: 1.The activity of INS-1 cells treated with different concentrations of drugs was compared by CCK-8 method.The concentration of SRL was 30 μg/m L,MET was 40 μg/m L and DAPA was 25 nmol/m L;2.Compared with VH group,the activity of INS-1 cells in SRL group was significantly decreased(P<0.05),the level of GSIS was decreased(P<0.05),the production of ROS was increased(P<0.05),the apoptosis was increased,and the protein expressions of Mn SOD,p-AMPK and p-FOXO-1 were decreased,while the protein expressions of Casepase-1,PI3 K and p-Akt were increased.SRL can induce injury of INS-1 cells and decrease insulin secretion function of INS-1 cells;3.Compared with SRL group,the activity of INS-1cells in single-drug treatment group(SRL+DAPA and SRL+MET groups)was significantly increased(P<0.05),the level of GSIS was increased(P<0.05),the production of ROS was decreased(P<0.05),the cell apoptosis was decreased,and the protein expressions of Mn SOD,p-AMPK and p-FOXO-1 were increased.The protein expression of Casepase-1,PI3 K and p-Akt decreased;4.There was no significant difference between SRL+DAPA group and SRL+MET group;5.Compared with single-drug treatment group(SRL+DAPA and SRL+MET group),INS-1 cell activity was significantly increased(P<0.05),GSIS level was further improved(P<0.05),ROS expression was decreased(P<0.05),the apoptosis were decreased,and the protein expressions of Mn SOD,p-AMPK and p-FOXO-1 were increased in SRL+MET+DAPA group.The protein expression of Casepase-1,PI3 K and p-Akt decreased.Conclusion: 1.MET combined with DAPA had a protective effect on SRL-induced INS-1 cell injury;2.DAPA is recommended as a second-line drug for the treatment of new-onset diabetes after transplantation on the basis of MET therapy.