Investigation Of The Mechanism Of Low-level Sodium Arsenite Induced Apoptosis Through Inhibition Of TrxR Activity In Pancreatic β-cells

Objective: Exposure to high concentration of arsenic has become a major public health problem all around the world. Arsenic is one of the most common contamination in groundwater. In China, nearly 20 million people might be exposed to the threat of high arsenic drinking water. High levels of arsenic in drinking water are reported to be associated with an increased risk of diabetes. It suggest that arsenic may have a toxic effect on islet cells. In all types of diabetes, type 2 diabetes(T2DM) accounted for more than 90% to 95%. Recently, epidemiological and experimental studies show that exposure of arsenic is an important risk factor for T2 DM. Mechanisms of arsenic-induced diabetes may be destruct pancreatic β cell function, and induced the islet β cell apoptosis. But the mechanism is not clear yet. To further investigate the relationship between arsenic and diabetes and its possible mechanism, we investigated the mechanism that low concentration of sodium arsenite inhibited thioredoxin reductase activity to induced apoptosis in rat pancreatic β cell line(INS-1).Methods: In this study, rat islet β cells(INS-1) were selected for the study. 3-(4, 5– Dimethylthiazol- 2- yl)- 2, 5- diphenyltetrazolium bromide(MTT) was used todetect the toxic effects of different concentrations of sodium arsenite on INS-1 cells.The apoptosis induced by different arsenic is detected with caspase-3 colorimetric assay; Endpoint insulin assay was used to detect the intracellular the activity of thioredoxin reductase(TrxR); Enzyme-linked immunosorbent assay(ELISA) was used to detect the level of intracellular apoptotic signaling kinase 1(ASK1); Western blot was used to detect the intracellular protein level of Bcl-2 associated X protein(Bax) and B-cell lymphoma/leukemia-2(Bcl-2) and the ratio of Bax/Bcl-2; The cell apoptosis induced by arsenic is detected with annexin V-FITC / PI flow cytometry assay.. SPSS 17.0 statistical software was used for statistical analysis.Results: After treatment with sodium arsenite(0 μM, 0.1 μM, 0.25 μM, 0.5 μM and 1.0 μM) for 96 h, sodium arsenite caused a dose-dependent decrease in cell viability in INS-1 cells; sodium arsenite caused a dose-dependent increase in the activity of caspase-3; sodium arsenite caused a dose-dependent decrase in the activity of thioredoxin reductase; the level of apoptosis signal kinase 1(ASK1) in INS-1 cells was increased in a concentration-dependent manner; Bax was up-regulated and anti-apoptic protein Bcl-2 was down-regulated; the ratio of Bax/Bcl-2 was accelerated after treatment with sodium aresenite; Knockdown of ASK1 attenuated sodium arsenite-induced apoptosis as shown in AV/PI staining.Conclusion: Sodium arsenite might inhibite the activity of TrxR and increase the level of ASK1 and the ratio of Bax/Bcl-2, and increase the activity of caspase-3subsequently. Finally, sodium arsenite caused apoptosis in INS-1 cells.