Protective Effect And Possible Mechanism Of Exendin-4 On Apoptosis Induced By Oxidative Stress In Murine Pancreatic β-cells

OBJECTIVE: To investigate the effects of tert-butyl hydroperoxide (t-BHP) on cell apoptosis in murine MIN6 pancreaticβ-cells and the changes in signaling pathway of NF-κB, iNOS and nitric oxide in this process. Further, to explore the effect and possible mechanism of Exendin-4 onβ-cells apoptosis induced by t-BHP.METHODS: Murine MIN6 pancreaticβ-cells were cultured in vitro and the cell apoptosis model of oxidative stress was established by tBHP. Cells were pretreated with Exendin-4 at the concentration of 10 and 100 nmol/L, or N-Acetyl-L-cysteine (NAC) and NG-Nitro-L-arginine Methyl Este (L-NAME) for 18h prior to tBHP exposure. Cell viability was measured by MTT assay. The morphological changes of cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-Ⅴ-FITC-PI staining. Nitric oxide levels was measured by Griess reagent assay. Inducible nitric oxide synthase(iNOS) protein and NF-κBp65 fragment were detected by Western blot.RESULTS: MIN6 cells viability were significantly reduced in a time-dependent and a dose-dependant manner after exposure of MIN6 cells to tBHP at range of 12.5~200μmol/L for 15~60min. The percentage of cell apoptosis was significantly increased after treating with tBHP. However, the exposure of tBHP at the concentration of 25μmol/L to MIN6 cells for 60min attained the maximal percentage of cells apoptosis but not at higher concentration. The higher concentration (over 100μmol/L) of tBHP-induced cells damage was shown as the death process shifted from apoptosis to necrosis. The levels of nitrite was elevated in a time-dependent manner in the process of apoptosis occurrence. Meanwhile, treatment of MIN6 cells with 25μmol/L tBHP produced the maximal response level of cytoplasmic iNOS protein at 30min, as well as nucleus NF-κBp65 fragment at 20min. Both L-NAME (500μmol/L) and NAC(10mmol/L) pretreatment significantly attenuated the elevated levels of nitrite and percentage of apoptosis due to tBHP alone(P<0.001). Intriguingly, Exendin-4 decreased both the increased levels of nitrite and percentage of apoptosis induced by tBHP in MIN6 cells in a dose-dependent manner(P<0.05). Forthermore, the present study showed that Exendin-4 patently reduced the expression of iNOS protein(P<0.01) and the ratio of nucleus:cytosol on NF-κBp65 proteins(P<0.001) induced by tBHP in MIN6 cells, which is similar to L-NAME or NAC, respectively.CONCLUSION: The cell signalling pathway of NF-κB-iNOS-nitric oxide mediated tBHP-induced apoptosis in MIN6 pancreaticβ-cells. Exendin-4 protectedβ-cells from tBHP-induced apoptosis via down-regulation of NF-κB-iNOS pathway. Glucagon-like peptide-1 receptor agonist—Exendin-4 is potentially a highly effective therapeutic agent in protecting pancreaticβ-cells.