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

目的探讨KATP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KATP阻断剂格列美脲(10μmol/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(10μmol/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APP695mRNA和APP695蛋白的表达(P <0. 05),并能降低细胞外液Aβ1-42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。

Objective To investigate the effects of ATP-sensitive potassium channel(K ATP)opener nicorandil on oxidative stress and production of amyloid-βprotein 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(10μmol/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-3βpathway 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).Aβ1-42 level in the medium was reduced either(P<0.05).In addition,nicorandil increased p-Akt and p-GSK-3βexpression by PI3K activation(P<0.05),which can be blocked by LY294002.Conclusion All these findings suggested that nicorandil might reduce the generation of Aβin APPsw/SH-SY5Y cells by inhibiting oxidative stress and activating PI3K/Akt/GSK-3βpathway.