摘要
目的探讨激活核因子红系2相关因子2(nuclear factor erythroid-derived 2-related factor 2,Nrf2)对1-甲基-4-苯基吡啶离子(MPP+)诱导PC12细胞氧化应激损伤的保护作用及其机制。方法以MPP+损伤PC12细胞作为帕金森病(PD)细胞模型,采用四甲基偶氮唑盐(MTT)法检测细胞存活率,流式细胞术检测细胞凋亡率,双氯荧光黄乙酸乙酯(DCF-DA)染色检测细胞内活性氧簇(ROS)的生成量,罗丹明123(Rh123)染色检测细胞线粒体膜电位(ΔΨm),免疫印迹法检测磷酸化的蛋白激酶B(pAkt)表达水平,评价Nrf2激动剂莱菔硫烷(SFP)对该损伤模型的影响并探讨其相关机制。结果①500μmol/L的MPP+可以导致PC12细胞的存活率下降,凋亡率增加,0.1、0.5、1.0、5.0、10.0μmol/L浓度的SFP可以使PC12细胞的存活率增加、凋亡率降低,并在5.0μmol/L时达到最大保护效果;②MPP+导致PC12细胞ROS生成增加、ΔΨm降低,SFP可以减轻该氧化应激损伤;③SFP增加Akt磷酸化水平,该效应及其抗氧化损伤作用可被PI3K抑制剂LY294002所拮抗。结论 Nrf2活化剂SFP能抑制MPP+对PC12细胞的氧化应激损伤,其机制是通过PI3K/Akt通路实现的。
Objective To investigate the protective effects of the Nrf2 agonist sulforaphane（SFP） on the MPP-induced in- jury of PC12 cells and the related mechanisms. Methods A cell model of Parkinson＇s disease was established by treating rat pheochromocytoma（PC12） cells with MPP＋. The cell viability was determined by MTT assay and the apoptosis rate of PC12 cells by flow cytometry. The cellular ROS level and the mitochondrial transmembrane potential were measured by spectroflu- orometry. The expression of pAkt in PC12 cells was detected by Western blot. Results The MPP＋ treatment reduced the via- bility and increased the apoptosis of PC12 cells, which could be reversed by SFP. And there occurred the maximum protective effect of SFP at a concentration of 5.0 μmol/L. Moreover,SFP could inhibit the production of ROS and the disruption of mito- chondrial transmembrane potential induced by MPP＋. SFP could increase the level of phosphorylation of Akt, a substrate of PI3K. It was also found that the anti-oxygen stress effect of SFP could be eliminated in the presence of the PI3K inhibitor, LY294002. The effect of SFP on the ROS levels and cell viability could also be blocked by LY294002. Conclusion The Nrf2 ag- onist sulforaphane may attenuate the MPP＋-induced oxidative stress in the PC12 cells through the PI3K/Akt-mediated signa- ling pathway.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2013年第2期143-147,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金青年科学基金资助项目(No.81100834)
