地塞米松诱导小鼠MIN6胰岛β细胞凋亡及其对AKT磷酸化的影响
Dexamethasone-induced apoptosis of murine MIN6 pancreatic β-cells and its effect on AKT phosphorylation
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摘要:
为观察地塞米松对小鼠MIN6胰岛β细胞凋亡及其对凋亡相关蛋白表达和AKT磷酸化的影响, 选用小鼠MIN6胰岛β细胞为研究对象, 用不同浓度地塞米松 (50、100、200、400及800 nmol·L−1) 诱导刺激不同时间, 观察细胞凋亡的变化。通过Hochest/PI、Annexin V-FITC细胞凋亡检测试剂盒观测细胞凋亡率; caspase-3检测试剂盒测定caspase-3含量; Western blotting测定细胞色素c (Cyt-c)、Bcl-2、Bax、T-AKT及p-AKT的蛋白含量。结果显示: 地塞米松 (50~800 nmol·L−1) 作用48 h后, 可诱导β细胞凋亡; 100 nmol·L−1地塞米松处理 72 h后, caspase-3含量明显增多; 不同浓度地塞米松处理48 h, 随着浓度的增加Cyt-c表达增多, Bax蛋白含量无明显变化, 而Bcl-2蛋白含量有所降低。T-AKT表达无明显变化, p-AKT随地塞米松浓度增加表达减少。因此, 地塞米松能够引起小鼠MIN6胰岛β细胞凋亡, 其机制可能与抑制AKT磷酸化、下调Bcl-2表达有关。
Abstract:This study is to investigate the effect of dexamethasone on cell apoptosis of murine MIN6 pancreatic β-cells, and to investigate the mechanism of dexamethasone-dependent cell apoptosis. The cell apoptosis model was established by choosing the murine MIN6 pancreatic β-cells, which was cultured in vitro and induced by dexamethasone. The morphology of the cell apoptosis was observed through fluorescence microscopic analysis after Hochest/PI staining and flow cytometric assay after Annexin-V/PI staining. The expression of caspase-3 was detected with caspase-3 activity assay kit. The expressions of Cyt-c, Bcl-2, Bax, AKT and p-AKT were observed with Western blotting. The results indicated that after exposure to dexamethasone at a concentration ranging from 50−800 nmol·L−1 for 48 h, the percentage of cell apoptosis was significantly increased with the concentration over 100 nmol·L−1 of dexamethasone; after exposure to dexamethasone (100 nmol·L−1) for 72 h, the activity of caspase-3 increased significantly; after exposure to dexamethasone at a concentration ranging from 50−800 nmol·L−1 for 48 h, the expression of Cyt-c increased, Bcl-2 and AKT phosphorylation decreased while Bax and T-AKT remained unchanged. It could be concluded that the effect of dexamethasone on murine MIN6 pancreatic β-cells apoptosis is significant. The mechanism of dexamethasone-dependent cell apoptosis is probably related to down regulation of the Bcl-2 expression and reduction of AKT phosphorylation.
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