铜、铁、锌、铝通过Akt/GSK-3β信号通路诱导SH-SY5Y细胞的凋亡

摘要

目的 探究铜、铁、锌、铝对人神经母细胞瘤细胞(SH-SY5Y)的神经毒性作用和机制. 方法 采用50、200、400 μmol/L CuSO4,1、2、4 mmol/L FeSO4和AlCl3,50、100、200 μmol/L ZnSO4分别作用于SH-SY5Y细胞,通过噻唑蓝(MTT)法检测细胞生长活性.采用ELISA法检测细胞内Akt、磷酸化Akt(Ser473)、GSK-3β及磷酸化GSK-3β(Ser9)蛋白的表达.采用Annexin-V/PI双染法检测细胞凋亡率. 结果 铜、铁、锌、铝能明显抑制细胞活性,并呈剂量依赖性,与对照组比较差异有统计学意义(P<0.05).铁和铝各剂量组Akt表达较对照组降低(P<0.05);各剂量组GSK-3β表达无明显差异(P>0.05);各剂量组Akt及GSK-3β的磷酸化水平低于对照组(P<0.05).细胞凋亡率随染铜、铁、锌、铝剂量的加大而升高(P<0.05). 结论 铜、铁、锌、铝可引起神经细胞毒性,抑制Akt活性,激活蛋白激酶GSK-3β,导致细胞凋亡.%Objective To explore the effects of copper, iron, zinc and aluminum on the neurotoxi-city and mechanism in SH-SY5Y cells.Methods SH-SY5Y cells were treated with different concentrations of copper sulfate (50,200,400μmol/L),iron sulfate and aluminum chloride (1,2,4 mmol/L),and zinc sulfate(50,100,200 μmol/L) respectively.Cell viabilities were measured by MTT assay.ELISA kit method were performed to evaluate the deliverances of glycogen synthase kinase-3β(GSK-3β), phosphorylation of the sites in GSK-3β-Ser9, Akt and phosphorylated Akt(Ser473).The apoptosis rate of detected cells was by double-staining with Annexin-V and PI.Results With the increase of the concentration of copper, iron, zinc and aluminum increased, the viability of SH-SY5Y cell decreased gradually.The cell viability of each dose group were significantly lower than that of control group(P<0.05).The expressions of Akt were significantly lower in iron and aluminum group, than that in control group (P<0.05).The expression of GSK-3β had no significant difference(P>0.05).The levels of phosphorylation of GSK-3β and Akt in all groups were significantly lower than those of control group(P<0.05).With the concentration of copper, iron, zinc and aluminum increased, the apoptosis rates were significantly higher than that of control group (P<0.05).Conclusions Copper, iron, zinc, aluminum could induce neurotoxicity, inhibit the activity of Akt, activate the glycogen synthase kinase 3β and lead to the neuronal apoptosis.