硫辛酸对高糖诱导小鼠RIN-m5F细胞保护作用的机制

Cytoprotective Effect of α-Lipoic Acid on RIN-m5F Cultured with High Glucose

摘要:

探究α?鄄硫辛酸(α?鄄LA)对高糖诱导RIN?鄄m5F细胞的保护机制。RIN?鄄m5F细胞分为对照组(11 mmol/L 葡萄糖)、葡萄糖损伤组(22 mmol/L或 44 mmol/L葡萄糖)、α?鄄 LA干预组(上述3个葡萄糖浓度+200 μmol/L α?鄄LA)，分别检测细胞内活性氧自由基(ROS)、丙二醛(MDA)水平、总抗氧化能力(T?鄄AOC)及超氧化物歧化酶(SOD)活性；BAX、BCL?鄄2、GSK?鄄3β mRNA表达水平；细胞线粒体膜电位及蛋白印迹法检测GSK?鄄3β和p?鄄Ser9 GSK?鄄3β蛋白质水平。结果表明，与糖损伤组相比，200 μmmol/L α?鄄LA显著降低胞内ROS水平，分别降低30%和83%；增强细胞SOD活性及T?鄄AOC能力，降低MDA积累；上调抗凋亡因子BCL?鄄2 mRNA表达水平，下调促凋亡蛋白质BAX mRNA表达水平，提高细胞线粒体膜电位。此外，α?鄄LA能够下调GSK?鄄3β mRNA表达水平，提高p?鄄Ser9 GSK?鄄3β蛋白质水平，表明α?鄄LA对高糖诱导RIN?鄄m5F细胞的抗氧化和抗凋亡保护作用与调节GSK?鄄3β有关。

Abstract:

The aim of this study is to investigate whether α-lipoic acid(α- LA) prevents high glucose-induced oxidative damage and apoptosis of pancreatic islet beta cells. In the present sudy，RIN-m5F cells were incubated with different concentration of glucose(11 mmol/L，22 mmol/L and 44 mmol/L) in the presence or absence of 200 mol/L α-LA for 72 h. Then，cellular reactive oxygen species(ROS) levels，malandialdehyde(MDA) levels，total antioxidant capacity(T-AOC) and superoxide dismutase(SOD) activity were assayed with the appropriate test kits；mitochondrial membrane potential was detected by flow cytometry；relative genes levels were analyzed by reverse transcriptase polymerase chain reaction；western blotting was used to determine protein expression of GSK-3β and p-GSK-3β(Ser9). The results show that α-LA significantly reduced the MDA formation and inhibited the ROS production induced by high glucose，whereas increased T-AOC and SOD activity. Additionally，α- LA induced membrane depolarization and increased mitochondrial membrane potential. These effects were mediated by Bcl-2 associated X protein(BAX) and B-cell lymphoma-2(BCL-2) expression. Western blotting indicated that α-LA inhibited GSK-3β by increasing p-GSK-3β(Ser9) level. Therefore，our data suggest that α-LA can effectively attenuate high glucose-induced RIN-m5F cell oxidative damage and apoptosis，by mechanisms which probably involves the inactivation of GSK-3β by phosphorylation. These findings provide a new interpretation on the role of α-LA in the treatment of diabetes.