Cyclosporine A Impairs The Secretion Function Of Beta Cells Through Epac Pathway

Objective As the main anti immunologic rejection drugs to transplant patients,cyclosporin A(CsA)has been recognized as one of the risk factors for Post-transplantation diabetes mellitus(PTDM),which may be related to its molecular mechanism is target organ insulin resistance,apoptosis of insulin secreting cells,insulin vesica exocytosis process disorder and so on.Many arguments are controversial and require further elucidation.On the other hand,the related research of exchange protein directly activated by c AMP(Epac)is the hot point of insulin secretion research as well as new drug research.The interacts of Epac and c AMP plays an important role in the regulation of insulin secretion.The hypothesis of this study is that the molecular mechanism of CsA inhibiting insulin secretion involves the c AMP/ Epac pathway in pancreatic islet beta cells.Methods(1)The rat insulinoma cell line INS-1 832/13 cells and INS-1 cells were cultured in vitro;(2)Different concentrations of CsA(0.5 g/ml,1 g/ml,5 g/ml,8 g/ml,10 g/ml)was added to cells.After 24 h incubation,cell morphology was observed under an inverted microscope.(3)After 24 h and 48 h incubation,3-(4,5-dimethyl-2-thiazol yl)-2,5-diphenyl-2-H-tetrazoli M bromide(MTT)test was used to determine cytotoxicity of CsA.(4)Quantitative real-time polymerase chain reaction(q PCR)was applied to evaluate expression of genes including pdx1,ins1,ins2,bcl-2,caspase-3 respectively to analyze the effect of CsA on apoptosis,insulin gene expression of islet beta cell.(5)Glucose stimulated insulin secretion(GSIS)test was applied to evaluate the effect of CsA intervention on insulin secretion.(6)The changes of intracellular c AMP content after CsA intervention were detected by enzyme linked immunosorbent assay(ELISA).(7)The cell protein was extracted after intervention,and the expression of Epac protein,CREB and P-CREB protein and apoptosis related protein(Caspase-3,Bcl-2)in INS-1 cells were detected by Western Blot.Results(1)After 24 hours incubation with CsA,INS-1 832/13 cells and INS-1 cells were observed under the inverted microscope.There almost no change in cell morphology at low concentrations of CsA(0.5 μg/ml,1 μg/ml);cells began to deteriorate at 5 μg/ml CsA concentration;at high concentration(8 μg/ml,10 μg/ml)CsA the cell state were bad,significant changes occurred in the morphology,cells turned to be shrinkage、round and light,floating cells and cell debris were increased.(2)After 24 hours incubation with CsA in INS-1 832/13 cells,MTT showed OD values of lower concentrations of CsA intervention group(0.5 μg/ml,1 μg/ml)were not changed compared with the control group,a relatively high concentration CsA intervention group(5 μg/ml,8 μg/ml,10 μg/ml)compared with the control group,the OD value decreased by 5%,12%,23%;after 48 hours incubation with CsA in INS-1 832/13 cells,OD values of low concentration CsA intervention group(0.5 μg/ml,1 μg/ml)were not affected compared with the control group,relative high concentration CsA intervention group(5 μg/ml,8 μg/ml,10 μg/ml)compared with the control group,the OD value decreased by 16%,29%,60%.After 24 hours incubation with CsA in INS-1 cells,compared with the control group,the OD value of low concentration CsA intervention group(0.5 μg/ml,1 μg/ml)decreased,a relatively high concentration CsA intervention group(5 μg/ml,8 μg/ml,10 μg/ml)compared with the control group,the OD value decreased by 51%,57%,69%,48 hours compared with the control group the OD value decreased by 29%,43%,48% 77%,95%.(3)The q PCR experimental results showed the lower concentration(0.5 μg/ml,1 μg/ml)of CsA group and the control group ins1 and ins2 content of m RNA was no significant difference.Relatively high concentrations(5 μg/ml,8 μg/ml,10 μg/ml)CsA group had a decreased m RNA content compared with the control group,while pdx1 m RNA showed no significant change;(4)Insulin secretion test showed when the concentration of CsA was 5 μg/ml,intracellular insulin content decreased,CsA+GLP-1 intervention group basically reversed this change.The GSIS test showed: 5μg/ml CsA intervention group compared with NC group,basal insulin secretion were decreased,16.7 m M glucose stimulation of pancreatic insulin secretion cyclosporine intervention group(1 μg/ml,5 μg/ml)decreased by 43% and 57% respectively,5 μg /ml CsA+GLP-1 group had no significant difference compared with NC group,the GLP-1 intervention group was increased by 55%.(5)After 24 hours incubation with CsA in INS1 cells,compared with the CsA group and control group,bcl-2 gene is slightly reduced;caspase-3 gene showed obvious up trend,but statistically difference occurred only in the concentration reached in 8 μg/ml and 10 μg/ml.(6)The results of Western blot showed that there was no significant difference in the apoptosis related protein Bcl-2,Caspase-3 protein occurred statistically difference in the concentration reached in 8μg/ml and 10 μg/ml.(7)Intracellular c AMP content was significantly decreased in cells after the intervention of CsA,and the effect was significantly improved after the intervention of GLP-1.(8)The results of Western blot showed that the Epac protein was significantly reduced in CSA group,but the intervention of CsA had no significant effect on P-CREB/CREB.Conclusions CsA damage to the islet cells in a dose and time-dependent manner,the effects of CsA on islet beta cell function may be related to cell lines,the sensitivity of INS-1 cells to CsA is stronger than INS-1 832/13 cells,in the low concentration of CsA cells 48 hours after the intervention group also showed obvious inhibition of cell activity.The c AMP/Epac pathway may be involved in the damage mechanism of CsA on pancreatic islet cells.GLP-1 can reverse the damage of cyclosporine by this mechanism.The c AMP/CREB pathway may not play a major role in the mechanism of CsA damage to pancreatic islet cells.