Cyclosporin A-Induced Gene Differential Expression Of NIT-1β Cells And Related Molecular Mechanism

Cycloporin A (CsA) is widely used for therapeutic purposes for rejection after organ and cell transplantations and autoimmune diseases as a kind of potent immunosurpressant. But recently, quite a lot of clinical observations revealed that CsA has some side-effects on pancreatic ?cells such as impaired glucose tolerance and can even lead to diabetes mellitus. So, to study and analyze the effects and mechanism of the effect of CsA on pancreatic ? cells is of great importance for the rational drug usage and prevention of the side-effects of CsA.Objective: To observe the impact of CsA on the insulin release of pancreatic ? cells. And to investigate the gene differential expression of NIT-1 cells treated by CsA at different time intervals. Then to explore the possible mechanism of the effects of CsA on insulin release combined with Bioinformatics.Methods: NIT-1 cells were exposed to CsA at various concentrations(0.05.. 0.1. 0.5. 1. 5.10 礛/L) at different time intervals(24h, 48h and 72h). And the insulin release was investigated. Differential gene expression was observed and analyzed using DNA microarrays after NIT-1 cells was treated with CsA (lOMm/L) for 24h and 48h respectively. The DNA microarray results were conformed by RT-PCR with 6 differentially expressed genes. We analyzed the differentially expressed genes by bioinformatic analysis and finally screened out 20 genes that have close relationships with CsA's effects on NIT-1 cells, and explored their related molecular mechanisms.Results:1. CsA can inhibit insulin release, and its inhibitory effects are concentration-dependent and time-dependent2. By DNA microarray hibrization and analysis, 84 genes were differentially expressed at 24h. Among them, 46 genes were down-regulated and 38 genes wereup-regulated. 710 genes were differentially expressed at 48h. Among them, 366 genes were down-regulated and 344 genes were up-regulated.3. We screened out 20 genes (functions already known) that exhibit close relationship to CsA's effects on NIT-1 cells. All of them were down-regulated. These genes include: genes related to oxidative phosphorylation (15 genes, Cox7al . Cox7c. Cox8a, Atp6vOe. Atp6vlgl. Atplb3, Atp5k. Atp5o, Mrps21 . Mrpl23, Mrpl36. Mrp64. Mrpl3. Mrpll3. Mrpll9), SNARE protein related genes (Stxbpl. Vamp4), Ca target protein (similar to CaM kinase II beta) gene, G protein related gene (Arl4), and Pax6. We explored their related molecular mechanisms with respect of CsA's inhibitory effects.onclusion: CsA can inhibit insulin release of NIT-1 pancreatic P cells. Its inhibitory effects are probably related to its inhibition of genes expression of some functional enzymes and proteins in the process of metabolization-secretion couplet mechanism such as oxidative phosphorylation and insulin exocytosis.