Effect of iNOS and NF-κB gene silencing on β-cell survival and function

Abstract

Purpose: Type I diabetes results from β-cell death and dysfunction, induced by infiltration of immune cells and local production of inflammatory cytokines. Therefore, we investigated the effect of iNOS and NF-κB gene silencing on β-cell survival and function.Methods: Rat insulinoma INS-1E cells were transfected with chemically synthesized siRNA after complex formation with Lipofectamine 2000. Cells were then treated with a cocktail of inflammatory cytokines (IL-1β+ TNF-α+ IFN-α), and glucose stimulated-insulin response and viability were determined. iNOS and NF-κB gene expression was assessed at mRNA level by real time RT-PCR. The effect of gene silencing was also correlated with cytokine-induced NO production and apoptosis.Results: Transfection of INS-1E cells with siRNAs silenced iNOS and NF-κB gene expression and reduced NO production in a sequence-specific manner without causing significant loss of cell viability and function. However, the abrogation of NO production did not prevent INS-1E cells from cytokine-induced apoptosis, suggesting that this event may not be totally dependent on NO production.Conclusion: The gene silencing approach presented here is capable of attenuating the effects of inflammatory cytokines, such as iNOS expression and NO production and it will help to identify new target genes to improve islet transplantation.