丙泊酚调节MPP+诱导的SH-SY5Y细胞线粒体氧化应激和凋亡

Propofol Regulates MPP+-induced Mitochondrial Oxidative Stress and Apoptosis in SH-SY5Y Cells

目的探讨不同浓度的丙泊酚(propofol,PPF)对1-甲基-4-苯基吡啶离子(1-Methyl-4-phenylpyridinium,MPP+)诱导的帕金森(Parkinson’s disease,PD)模型细胞氧化应激和凋亡的作用。方法利用1 mM MPP+诱导人神经母细胞瘤细胞SH-SY5Y构建PD细胞模型。PPF处理组分别用10、20、40、80μM PPF在MPP+诱导前刺激SH-SY5Y细胞4 h。细胞计数法(cell counting kit-8,CCK-8)评估细胞增殖活力。2′,7′-二氯二氢荧光素二乙酸酯(H2DCF-DA)荧光探针用于检测细胞中活性氧(reactive oxygen species,ROS)。试剂盒分别检测丙二醛(malondialdehyde,MDA)和还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced nicotinamide adenine dinucleotide phosphate oxidase,NADPH)氧化酶的水平。Western blot用于检测线粒体和细胞质中细胞色素c的蛋白表达以及细胞中Bcl-2、Bax和Cleaved caspase-3蛋白表达。流式细胞术分析细胞凋亡率。结果MPP+诱导明显抑制SH-SY5Y细胞的增殖(P<0.001),促进细胞中ROS(P<0.001)、MDA(P<0.001)、NADPH氧化酶(P<0.01)、细胞质中细胞色素c的表达(P<0.01)并诱导细胞凋亡(P<0.001)以及促凋亡蛋白Bax和Cleaved caspase-3的表达(P<0.01),减少线粒体中细胞色素c的蛋白表达(P<0.01)和抗凋亡蛋白Bcl-2的表达(P<0.01)。PPF预处理可缓解MPP+诱导引起的SH-SY5Y细胞增殖抑制以及氧化应激和凋亡的促进作用(P<0.001),且40μM和80μM对细胞的作用更为显著。结论PPF预处理可缓解MMP+诱导的SH-SY5Y细胞氧化应激,减少细胞凋亡。

Objective To investigate the effects of different concentrations of PPF on oxidative stress and apoptosis of PD model cells induced by MPP+.Methods The human neuroblastoma cell SH-SY5Y was induced by 1 mM MPP+to establish PD cell model.In PPF treatment group,SH-SY5Y cells were stimulated with 10,20,40 and 80μM PPF for 4 h before MPP+induction.Cell counting kit-8(CCK-8)was performed to evaluate cell proliferation activity.H2DCF-DA fluorescent probe was used to detect ROS in cells.The levels of MDA and NADPH oxidase were analyzed by the kit.Western blot examined the protein expression of cytochrome c in mitochondria and cytoplasm,as well as the relative expression of Bcl-2,Bax and cleaved caspase-3 in SH-SY5Y cells.Apoptosis rate was analyzed by flow cytometry.Results MPP+significantly inhibited the proliferation of SH-SY5Y cells(P<0.001),promoted the level of ROS(P<0.001),MDA(P<0.001),NADPH oxidase(P<0.01),cytochrome c in cytoplasm(P<0.01)and induced apoptosis(P<0.001)and the relative expression of pro-apoptosis protein Bax and cleaved caspase-3(P<0.01),reduced cytochrome c protein in mitochondria(P<0.01)and the relative expression of anti-apoptosis protein(P<0.01).PPF pretreatment alleviated the proliferation inhibition,oxidative stress and apoptosis promotion of SH-SY5Y cells induced by MPP+(P<0.001),and the effects of 40μM and 80μM on cells were more significant.Conclusion PPF pretreatment can alleviate the oxidative stress of SH-SY5Y cells induced by MMP+and reduce apoptosis rate.