DU S-C, Ge Q-M, Lin N, Dong Y, Su Q
Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Department of Endocrinology, Shanghai, China.
Cell Mol Biol (Noisy-le-grand). 2012 Mar 23;58 Suppl:OL1654-9.
Overproduction of reactive oxygen species (ROS) or exhaustion of antioxidants may cause oxidative stress which is a major factor of defective insulin secretion and increases apoptosis of pancreatic β-cells in diabetes. So there comes a consideration of whether antioxidant strategies can be used to protect deterioration of the β-cells. In this study, we explored the mechanism of oxidative stress mediated lipopolysaccharide (LPS) induced apoptosis in insulin secreting (INS-1) cells from a rat pancreatic β-cell line. ROS was monitored by using intracellular ROS capture dihydroethidium (DHE) and dihydrorhodamine123 (DHR123). Apoptosis rate was measured by flow cytometry (FCM). The pro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 were analysed by Western blot and RT-PCR. The results demonstrate that LPS-stimulated INS-1 cells manifest intensified intracellular fluorescence in both dose- and time- dependent manners. Apoptosis rate of LPS stimulated INS-1 cells is significantly increased by FCM, with a significant increase in Bax/Bcl-2 ratio revealed by Western blot and RT-PCR. Furthermore, α-lipoic acid (α-LA) inhibits LPS-induced apoptosis, but can not restore the function of glucose stimulated insulin secretion (GSIS) in INS-1 cells.
活性氧（ROS）的过度产生或抗氧化剂的耗尽可能会导致氧化应激，这是胰岛素分泌缺陷的一个主要因素，并会增加糖尿病患者胰腺β细胞的凋亡。因此，人们开始考虑抗氧化策略是否可用于保护β细胞的退化。在本研究中，我们从大鼠胰腺β细胞系胰岛素分泌（INS-1）细胞中探索了氧化应激介导的脂多糖（LPS）诱导凋亡的机制。通过使用细胞内ROS捕获剂二氢乙锭（DHE）和二氢罗丹明123（DHR123）来监测ROS。通过流式细胞术（FCM）测量凋亡率。通过蛋白质印迹法和逆转录-聚合酶链反应（RT-PCR）分析促凋亡基因Bax和抗凋亡基因Bcl-2。结果表明，LPS刺激的INS-1细胞在剂量和时间依赖性方式下均表现出细胞内荧光增强。FCM显示LPS刺激的INS-1细胞凋亡率显著增加，蛋白质印迹法和RT-PCR显示Bax/Bcl-2比值显著增加。此外，α-硫辛酸（α-LA）可抑制LPS诱导的凋亡，但不能恢复INS-1细胞中葡萄糖刺激的胰岛素分泌（GSIS）功能。
