叠氮钠、过氧化氢对SH-SY5Y细胞内硫氧还蛋白还原酶的影响

Effects of H2O2 or NaN3 on Thioredoxin Reductase in SH-SY5Y Cells

过度氧化应激是诱发许多神经退变病的重要因素。叠氮钠(NaN3)是线粒体有氧呼吸链细胞色素c氧化酶(COX)的特异性抑制剂,过氧化氢(H2O2)释放氧自由基造成氧化损伤,两者都可以用于氧化应激情况下神经元损伤模型的建立。硫氧还蛋白还原酶(thioredoxin reductase,TR)特异性的还原氧化型的硫氧还蛋白(thioredoxin,TRx),调节细胞中氧化还原的平衡。现以不同浓度NaN3或H2O2处理人神经母细胞瘤细胞(SH-SY5Y细胞),建立损伤模型。通过MTT法、形态学方法检测SH-SY5Y细胞损伤程度。同时,通过Western blot定量法、免疫细胞化学法,检测损伤的SH-SY5Y细胞中TR含量的改变,观察TR在胞内的分布。实验表明,NaN3、H2O2均以浓度依赖方式损伤SH-SY5Y细胞;TR分布于SH-SY5Y细胞的胞浆,表明TR是一种分泌蛋白,损伤后分布无明显变化。但一定浓度的NaN3作用后3h,胞内TR水平显著降低,即神经系统内呼吸链受损可抑制TR的表达,为神经退变病的防治提供了新的思路。

Increasing experimental evidence demonstrated a role of oxidative stress in the pathogenesis of neurodegeneration diseases. Sodium azide (NaN3) is a special inhibitor of mitochondrion cytochome c oxidase(COX), and H2O2 can produce lots of free redicals, so both of them can mimic neuronal injury induced by oxidative stress. In this experiment, the neurotoxic effects of H2O2 or NaN3 on SH-SY5Y cells were detected by means of cell viability measurement (MTT) and morphological observation. Moreover, the changes of intracellular thioredoxin reductase(TR), the enzyme responsible for reduction of oxidized thiredoxin (Trx), were analyzed through Western blotting and the intracellular distribution of TR was observed using immunohistochemical method. It was found that SH-SY5Y cells could be insulted by either NaN3 or H2O2 in a dose-dependent manner. TR was identified in plasma, suggesting it is a secreted protein. A substantial down-regulation of TR in SH-SY5Y cells was only observed 3 h later following NaN3-induced injury. This decrease in the antioxidative TR may contribute to the dysfunction of mitochondria and subsequent neurodegeneration.