过表达脑红蛋白对水溶性β-淀粉样蛋白片段1-42诱导SH-SY5Y细胞线粒体损伤的保护作用及其机制

Protective role of over-expression of neuroglobin in mitochondrial injury of SH-SY5Y cells induced by water-soluble amyloid-beta 1-42 and relevant mechanisms

目的观察过表达脑红蛋白(neuroglobin,NGB)对水溶性β-淀粉样蛋白片段1-42(Amyloid-beta 1-42,Aβ_(1-42))诱导SH-SY5Y细胞线粒体损伤的保护作用,并探讨其机制。方法体外培养SH-SY5Y细胞,经Aβ_(1-42)预处理后转染质粒p EGFP-NGB及空载体p EGFP-N1,同时设空白对照组(未转染)。MTT法、流式细胞术及JC-1染色法分别检测过表达NGB对Aβ_(1-42)诱导损伤的SH-SY5Y细胞存活率、细胞凋亡及线粒体膜电位的影响,采用相应试剂盒检测细胞内半胱氨酸蛋白酶3(caspase 3)、caspase 9、ATP及细胞色素C(cytochrome C,cyto C)氧化酶活性;Western blot法检测细胞中cyto C、凋亡蛋白酶激活因子-1(apoptosis protease activating factor-1,Apaf-1)、caspase 3及caspase 9蛋白的表达水平。结果过表达NGB可显著提高Aβ_(1-42)诱导损伤SH-SY5Y细胞的存活率(P<0.001)及细胞中cyto C氧化酶、ATP的活性(P<0.01);可显著抑制损伤细胞的凋亡(P<0.05)、线粒体膜电位的降低(P<0.001)及细胞中caspase 3、caspase 9的活性(P<0.01);可明显降低损伤细胞内cyto C、Apaf-1、caspase 3及caspase 9蛋白的表达水平(P<0.05)。结论 NGB可显著抑制Aβ_(1-42)诱导的SH-SY5Y细胞损伤,促进其增殖,其机制可能与NGB恢复线粒体功能及抑制cyto C触发细胞凋亡密切相关。

Objective To observe the protective role of over-expression of neuroglobin（NGB）in the mitochondrial injury of SH-SY5 Y cells induced by water-solube amyloid-beta 1-42（Aβ_（1-42））and investigate the relevant mechanism. Methods SH-SY5 Y cells were cultured in vitro,pretreated with Aβ_（1-42） and transfected with plasmid p EGFP-NGB,using those transfected with empty vector p EGFP-N1 as parallel control and those untransfected as blank control. The effects of overexpression of NGB on the survival rate,apoptosis and mitochondrial membrane potential of SH-SY5 Y were evaluated by MTT assay,flow cytometry and JC-1 staining respectively,while the activities of cytochrome C oxidase,caspase 3,caspase 9 and ATP in the cells were determined by the corresponding kits,and the expression levels of cyto C oxidase,apoptosis protease activating factor-1（Apaf-1）,caspase 3 and caspase 9 by Western blot. Results Over-expression of NGB significantly increased the survival rate of injured SH-SY5 Y cells induced by Aβ_（1-42）（P 〈0. 001） as well as the activities of cyto C oxidase and ATP（P 〈0. 01）,while significantly inhibited the cell apoptosis rate（P 〈0. 05）,the decrease of mitochondrial membrane potential（P 〈0. 001） and activities of caspase 3 and caspase 9（P 〈0. 01）,and significantly decreased the expression levels of cyto C,Apaf-1,caspase 3 and caspase 9（P 〈0. 05）. Conclusion NGB may significantly inhibit the injury of SH-SY5 Y cells induced by Aβ_（1-42） and promote the cell proliferation by a mechanism which is possibly associated with recovering the function of mitochondria and inhibiting the apoptosis of cells triggered by cytochrome C.