尼可地尔对APPsw/SH-SY5Y细胞氧化应激和Aβ生成影响的研究

Effects of nicorandil on oxidative stress and Aβ production in APPsw/SH-SY5Y cells

关键词:  尼可地尔  阿尔茨海默病  氧化应激  β-淀粉样蛋白  PI3K/Akt/GSK-3β

Key words:  Nicorandil  Alzheimer’s disease  oxidative stress  β-amyloid protein  PI3K/Akt/GSK-3β

摘要:

目的　探讨K_ATP开放剂尼可地尔对APPsw/SH-SY5Y细胞氧化应激和β-淀粉样蛋白（Aβ）生成的影响及其相关机制。方法　APP695swe质粒瞬时转染SH-SY5Y细胞24 h后，尼可地尔（1 mmol/L）和K_ATP阻断剂格列美脲（10 μmol/L）分别或共同作用24 h，收集细胞进行生化，real-time PCR，western blot和 ELISA检测；PI3K抑制剂LY294002（10 μmol/L）预处理，检测p-AKT和p-GSK-3β蛋白的表达。结果　尼可地尔可以明显降低MDA的生成（P＜0.05），增加总SOD和GSH的水平（P＜0.05）；尼可地尔可以明显降低APP695 mRNA和APP695蛋白的表达（P＜0.05），并能降低细胞外液Aβ1-42的水平（P＜0.05）。尼可地尔可以明显上调p-AKT和p-GSK-3β蛋白的表达（P<0.05），并可被LY294002预处理所抵消。结论　尼可地尔可能通过抑制氧化应激，激活PI3K/Akt/GSK-3β通路等机制减少APPsw/SH-SY5Y细胞Aβ的生成。

Abstract:

Objective　To investigate the effects of ATP-sensitive potassium channel(K_ATP) opener nicorandil on oxidative stress and production of amyloid-β protein in APPswe/SH-SY5Y cells and the possible mechanisms involved. 　Methods　After SH-SY5Y cells were transiently transfected with APP695swe plasmids 24 h, cells were treated with nicorandil (1 mmol/L) for 24 h with and without glibenclamide (10 μmol/L), a K_ATP inhibitor. The cells were collected for biochemical assays, real-time PCR, western blot and ELISA assay. PI3K inhibitor LY294002 was applied to investigate the regulatory effect of nicorandil on PI3K/Akt/GSK-3β pathway in APPsw/SH-SY5Y cells. 　Results　Nicorandil increased the activity of total SOD and GSH as well as decreased the activity of MDA (P＜0.05). Moreover, nicorandil down-regulated APP695 expression on mRNA and protein level (P＜0.05). Aβ_1-42 level in the medium was reduced either (P＜0.05). In addition, nicorandil increased p-Akt and p-GSK-3β expression by PI3K activation (P＜0.05), which can be blocked by LY294002. 　Conclusion　All these findings suggested that nicorandil might reduce the generation of Aβ in APPsw/SH-SY5Y cells by inhibiting oxidative stress and activating PI3K/Akt/GSK-3β pathway.