O16 Activation of Nrf2 pathway by Compound A (CpdA) protects b-cells against cytokine-mediated inflammatory injury

Abstract

Introduction: The development of the autoimmune process during type 1 diabetes (DM1) contributes to insulitis; in this context, both β-cell stress and subsequent insulin secretory deficiency precede clinical signs of disease. Hyperglycemia triggers excess production of mitochondrial reactive oxygen species (ROS) that overwhelm the anti-oxidative capacity of β-cells, leading to oxidative stress. Islet inflammatory microenvironment during the autoimmune attack contributes to the activation of oxidative and endoplasmic reticulum (ER) stress resulting in β-cell dysfunction and death. The transcription factor Nrf2 regulates the expression of cytoprotective genes in response to oxidative stress, its induction is crucial for the normal β-cell physiology. We reported that Compound A (CpdA), a dissociative glucocorticoid receptor-ligand, is an effective modulator of key immune cells involved in insulitis, T and dendritic cells. In addition, we observed that CpdA ameliorates cytokine (IL-1b+IFN-g; CYT)-induced ER stress in β-cells and that in vivo CpdA administration leads to a significant delay of disease onset in an accelerated murine model of DM1. The development of new agents, with anti-inflammatory and immunomodulatory action and protective potential directed against dysfunctional signaling of β-cells is of clinical interest in diabetes.

Objectives: The aim of this study was to explore the effect of CpdA on Nrf2 signaling pathway and CYT-induced oxidative stress in β-cells.

Materials & Methods: A rat insulinoma cell line (INS-1E) was used as experimental model. CpdA chemical formula: 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride. A reporter plasmid (ARE-LUC) was used for the evaluation of Nrf2 transcriptional activity (luminometry); RTqPCR (Sybr Green) for analysis of mRNA expression; WB for protein expression analysis; a DCFDA based commercial kit to evaluate ROS (fluorometry); MTT assay for cell viability analysis and ELISA for insulin.

Results: CpdA (10μM) enhanced Nrf2 transcriptional activity and mRNA expression of
several target antioxidant enzymes (NQO1, HMOX-1 and Txnrd1) in INS-1E (p<0.05 vs.
control). Consequently, CpdA reduced CYT-induced ROS generation in INS-1E cells (p<0.01 vs. control). CpdA action counteracted the increase in the apoptotic index, quantified by Bax / Bcl-2 expression ratio, and in DP5 (death protein-5) mRNA expression, both parameters induced by CYT (p <0.05 vs. control). CpdA protected against the reduction of viability and enhanced basal insulin secretion in CYT-challenged INS-1E cells (p<0.05 vs. control).

Conclusions: CpdA treatment activates Nrf2 pathway decreasing oxidative stress and apoptosis and maintaining normal basal insulin secretion when β-cells are exposed in vitro to a CYT-induced inflammatory environment. Currently, several experiments are ongoing in our laboratory in order to elucidate CpdA therapeutic potential on autoimmune diabetes.