In this study, RAW264.7 cells were co-cultured with MIN6 cells to simulate the T1D pathogenic microenvironment, with the anti-oxidative enzyme peroxiredoxin-1 knocked down in one or both cell types in order to elucidate the function of this enzyme in the pathogenesis of T1D. The protective effect of Bcl-xL, an anti-apoptotic gene in cytokine-induced β-cell apoptosis has previously been reported [10
]. In our results, MIN6 cells co-cultured with LPS-stimulated shPRX-1 cells displayed increased expression of Bcl-xL as shown in after L-NMMA was treated and it seems that the apoptosis-inducing ability of shPRX-1 RAW264.7 cells are somewhat inhibited compared to scramble RAW264.7 cells. While when shPRX-1 MIN6 cells are coupled with scramble RAW264.7 cells, the expression of the pro-apoptotic gene Bim was shown to be significantly increased (). It seems that the knockdown of PRX-1 induces a certain vulnerability in MIN6 cells to RAW264.7 cell induced apoptosis. Supporting our findings, a recent study on peroxirodoxin-3, a family member of peroxiredoxin, showed that it protects pancreatic β-cells from apoptosis as well [11
The protective effects of IL-6 on pancreatic β cells have been reported by numerous investigators [12
]. As IL-6 is readily secreted by activated macrophages [15
], it was hypothesized that IL-6 produced by RAW264.7 cells may be the cause of increased expression of Bcl-xL in co-cultured MIN6 cells. Also the anti-inflammatory cytokine, IL-10 has been reported to accelerate destruction of β-cells in nonobese diabetic mice [16
]. However, in our results, while IL-6 decreased the production of NO in both scramble and shPRX-1 MIN6 cells under inflammatory cytokine stimulation, treatment IL-10 did not show any significant decrease in NO production (). Also, IL-6 treatment increased the viability of MIN6 cells under inflammatory conditions (). Interestingly, the protective effect of IL-6 was hindered in shPRX-1 MIN6 cells, suggesting a role for PRX-1 in preserving β-cell protective effects of IL-6. A similar trend was observed in gene expression levels of iNOS, Bcl-xL and Bim. The treatment with IL-6 increased the expression of the anti-apoptotic gene Bcl-xL, and decreased the expression of the pro-apoptotic gene Bim in scramble MIN6 cells ().
A recent study indicated that inhibition of the endogenous and exogenous IL-6 induced STAT-3 phosphorylation in human pancreatic cancer cells reduces cell viability [17
]. Therefore, the phosphorylation of STAT3, which is the principal mediator of IL-6 signaling, was observed through western blot in this current study. This was performed in order to determine whether PRX-1 function affects the protective effects of IL-6. Compared to scramble cells, shPRX-1 MIN6 cells were shown to have lower levels of STAT3 phosphorylation in response to IL-6 (). The results suggest that PRX-1 deficiency results in downregulation of STAT3 phosphorylation, and may consequently hinder cytoprotective effects of IL-6 in pancreatic β cells.
Overall, our results suggest that the deficiency or loss of activity of PRX-1 in pancreatic β-cells will result in decreased viability of these cells and may expedite the progression of type 1 diabetes. Along with the previously reported cytoprotective effects of this antioxidant enzyme, it seems that PRX-1 affects the protective action of IL-6 on pancreatic β cells. This suggests new roles for PRX-1 in immunological dysregulation. Future studies utilizing in vivo models would shed light on the clinical implications of this enzyme and may open new doors in the treatment of type 1 diabetes.