氧化应激在地塞米松诱导胰岛β细胞凋亡中的作用

EFFECTS OF OXIDATIVE STRESS ON THE APOPTOSIS OF PANCREATIC Β-CELLS INDUCED BY DEXAMETHASONE

目的通过观察地塞米松(Dex)对胰岛β细胞NADPH氧化酶表达水平和细胞凋亡的影响,探讨氧化应激在地塞米松诱导胰岛β细胞凋亡中的作用。方法将大鼠胰岛细胞β系INS-1细胞分为正常对照组、不同浓度的地塞米松组(0.01μΜ-Dex组、0.1μΜ-Dex组、1μΜ-Dex组)。培养48h后MTT法检测细胞增殖情况;Annexin V-FITC/PI双染经流式细胞仪检测细胞凋亡情况;Realtime-PCR检测Nox1、Nox3、Nox4、p47phox mRNA表达水平;荧光法检测ROS释放量;Weastern-blot检测Nox4蛋白表达情况。结果地塞米松诱导INS-1细胞凋亡呈现时间剂量依赖性,并且在此凋亡过程中Nox1、Nox3、Nox4、p47phox mRNA表达上调,Nox4蛋白表达上调,ROS释放量增加,差异均具有统计学意义(P<0.05)。结论地塞米松可诱导INS-1细胞凋亡,其机制可能与上调Nox1、Nox3、Nox4、p47phox表达,激活氧化应激反应有关。

Objective To investigate the role of oxidative stress in Dexamethasone （Dex） - induced pancreatic beta- cell death, we observed the effect of the Dex on the expression of NADPH oxidase and apoptosis of pancreatic beta cells. Methods The rat pancreatic β- cell line （INS- 1） was treated with Dexamethasone in a dose dependent manner （0.01, 0. 1, and 1μM）. After 48 hours of treatment, cell proliferation was estimated by MTT assay, and the occurrence of apoptosis was evaluated by Annexin V-FITC and propodium isodie （PI） double staining technique. In addition, we performed fluorometric assay to measure intracellular Reactive Oxygen Species （ROS） levels. The mRNA expression of Nox- 1, Nox- 4, and P47 was determined by Real - time PCR. Moreover, we also detected the Nox- 4 expression by Western blot analysis. Results Our data showed dose- and time- dependent effect of Dex on apoptosis of INS- l cells. Interestingly, the Nox- 1 and P47 mRNA expression levels were upregulated, and the Nox-4 was upreg-ulated at both mRNA and protein levels in apoptotic cells. Furthermore, ROS production was increased. The differences were statistically significant（ P〈0. 05 ）. Conclusion Collectively, these data suggest that treatment with Dex may cause apoptosis of INS- 1 cells, and enhanced Noxl, Nox3, Nox4, p47phox expression, which might be involve in the activation of oxidative stress.