Effects And Action Mechanism Of Cyclosporin A On NIT-1 Cells Proliferation And Insulin Secretion

Objective Cyclosporin A (CsA) is a potent immunosuppressive drug widely used for prevention of allograft rejection after organ transplantation, such as heart, kidney, liver, pancreas, and lungs. Furthermore, efforts have been made to delay the autoimmune process leading to insulin-dependent diabetes mellitus by early CsA therapy. In contrast, there are many reports pointing to significant adverse effects of CsA on β -cell function. Thus, one general problem in immunosuppressive therapy with CsA is the elevated incidence of impaired glucose tolerance or overt diabetes mellitus. The purpose of this dissertation is to investigate the effect of CsA on NIT-1 cells proliferation and insulin secretion in vitro and to find out the influence of CsA on the level of DNA polymerase a catalytic subunit 1 (Pol a 1) mRNA, mitochondrial oxidative phosphorylation enzymes (Nuox9,Nuox23,Uqcr,Cox7c,Atp5o and Atp5k) mRNA and mitochondrial ribosomal protein mRNA expression in NIT-1 cells by semi-quantitative reverse transcript polymerase chain reaction(RT-PCR) in order to discover the mechanism of the effects of CsA on cell proliferation and insulin secretion at gene level. Methods and results The present study is devided into 2 parts according to individual purpose.1. Effects of CsA on NIT-1 cells proliferation and the action mechanism at gene level NIT-1 cells were exposed to cyclosporin A at various concentrations(0.01 0.05, 0.1, 0.5, 1,5, 10μM)for 24h, 48h and 72h,cell proliferation was analyzed with MTT assay. The biphasic secretion of insulin was observed in response to CsA. In the case of incubation with CsA (≥5μM) for 24h, cell proliferation was promoted as compared with control. However, when incubated with CsA in various concentrations for 48h and 72h, cell proliferation was significantly impeded in a dose dependent manner. Incubation with l0μM CsA led to a maximal effects on cell proliferation, resulting in a complete inhibition of cell proliferation in the period between 48h and 72h. To further examine the effects of CsA on expression of Pol a 1 mRNA, the expression of Pol a 1 gene was assessed by semi-quantitative reverse transcription polymerase chain reaction in the presence or absence of l0μM CsA for 24h and 48h. The treatment of NIT-1 cells with l0μM CsA for 24h increased Pol a 1 mRNA expression. The promotion rate was 34.5%.This was in contrast to the substantial decrease of the gene expression when treated for 48h with an inhibition rate of 20.6%. These results were consistent to the effects of CsA on cell proliferation.2. Effects of CsA on insulin secretion from NIT-1 cells and the action mechanism at gene level NIT-1 cells were exposed to cyclosporin A at various concentrations(0.01, 0.05, 0.1, 0.5, 1, 5, l0μM) for 24h, 48h and 72h, cumulate insulin secretion was determined by using radioimmunoassay(RIA).The results showed that CsA inhibited cumulate insulin secretion in a dose dependent manner. To further investigate the effects of CsA on acute glucose-induced insulin secretion, we preincubated NIT-1 cells for 30min in KRBH buffer without glucose, and then examined insulin secretion from the cells with 3mM and 25mM glucose for Ih in the presence of CsA in KRBH buffer. In contrast to the previous study, treatment of NIT-1 cells with CsA for Ih in KRBH buffermarkedly increased basal and glucose-induced insulin secretion. To investigate the effects of CsA on expression of mitochondrial oxidative phosphorylation enzyme(Nuox9,Nuox23,Uqcr,Cox7c,Atp5o and AtpSk) genes expression and mitochondrial ribosomal protein (Mrpll3 and Mrps21) genes , the mRNA expression of genes was assessed by semi-quantitative reverse transcription polymerase chain reaction in the presence and absence of l0μM CsA for 24h and 48h. It was found that treatment of NIT-1 cells with lOuM CsA resulted dramatically in reduced oxidative phosphorylation enzyme (Nuox9, Nuox23, Uqcr, Cox7c, Atp5o and AtpSk) genes expression.