【作者】 肖海兵；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2011， 博士

【摘要】 目的本部分研究旨在帕金森病(Parkinson’s disease, PD)的PC12细胞模型中证实丙炔苯丙胺的神经保护作用,并对其保护机制进行初步探索。方法应用1-甲基-4-苯基吡啶离子(MPP+)处理PC12细胞制备PD的细胞模型。采用四甲基偶氮唑盐比色法(MTT法)、细胞乳酸脱氢酶(LDH)释放量检测、活性氧蔟(ROS)含量检测、四唑氮蓝/甘氨酸盐法行醌结合蛋白检测、Western Blots等方法检测丙炔苯丙胺对MPP+所致PC12细胞损伤的保护作用,初步探索丙炔苯丙胺是否通过间接的抗氧化作用发挥细胞保护效应。结果MTT法和LDH释放量检测均显示,不同浓度丙炔苯丙胺预处理可以显著减轻MPP+导致的PC12细胞损伤。ROS含量测定以及四唑氮蓝/甘氨酸盐法醌蛋白浓度检测的结果显示,不同浓度丙炔苯丙胺预处理明显地逆转了MPP+导致的氧化应激标志物增高。Western blots检测细胞内辅酶Ⅰ醌类氧化还原酶1(NQO1)蛋白表达及二氯靛酚反应法测定NQO1活性的结果显示,不同浓度的丙炔苯丙胺可以导致该抗氧化蛋白的表达量和酶活性均呈现出剂量依赖式的增高。另外,LDH释放量检测结果显示,双香豆素抑制NQO1后,丙炔苯丙胺对细胞的保护作用被逆转了。结论丙炔苯丙胺可以通过上调抗氧化蛋白NQO1的表达和活性,减少氧化应激损伤标志物的生成,发挥对MPP+所致的PC12细胞损伤的保护作用。目的验证丙炔苯丙胺是否通过促进核因子红系2相关因子2(Nrf2)核转位介导PC12细胞抗氧化蛋白NQO1表达和酶活性增高。方法应用细胞核蛋白与细胞浆蛋白抽提试剂盒分别提取PC12细胞的胞浆胞核蛋白,再采用Western blots技术检测胞浆胞核中Nrf2的蛋白含量变化;采用Nrf2小分子干扰RNA(SiRNA)转染技术,进一步验证Nrf2在丙炔苯丙胺诱导NQO1酶活性上调过程中的作用。结果Western blots结果表明,在MPP+的单独处理下,PC12细胞胞浆和胞核的Nrf2含量都出现了减少。而丙炔苯丙胺预处理可以有效地减少胞浆内的Nrf2含量,增加核内Nrf2的含量,且其胞浆内Nrf2的相对表达水平比MPP+单独处理时还要低,差异均有统计学意义。在转染了Nrf2 SiRNA的PC12细胞中,NQO1的活性出现了明显降低。另外,在Nrf2基因被沉默后,丙炔苯丙胺所导致的NQO1酶活性上调也消失了。结论丙炔苯丙胺所导致的抗氧化蛋白NQO1酶活性增强是通过促进Nrf2核转位,从而驱动NQO1转录增强这一途径完成的。目的本部分研究旨在探索丙炔苯丙胺促进Nrf2核转位的上游激酶调控通路,并观察丙炔苯丙胺对B型单胺氧化酶(MAOB)的抑制作用是否也参与了Nrf2的激活过程。方法应用Western Blots方法检测PC12细胞中Akt和细胞外信号调节激酶(Erk)等激酶的磷酸化水平变化,并应用各种激酶抑制剂处理PC12细胞观察各个激酶对Nrf2和NQO1的调控作用。另外,我们应用MAOB和Nrf2 SiRNA转染,来分析丙炔苯丙胺对MAOB抑制作用是否影响了Nrf2介导的抗氧化反应。结果经过Western blots法检测,我们发现,丙炔苯丙胺处理PC12细胞可以诱导Akt和Erk的磷酸化水平呈时间依赖性增高。另外,丙炔苯丙胺介导的Nrf2核转位和NQO1表达增高被可以被PD98059部分阻断,而PI3K的抑制剂LY294002几乎完全地取消了丙炔苯丙胺导致的Nrf2核转位和NQO1表达增高。对SiRNA转染的PC12细胞的ROS测定表明,MAOB基因沉默可以使ROS含量出现少量的下降,但并不影响丙炔苯丙胺的抗氧化活性。而且,无论MAOB基因沉默与否,Nrf2 SiRNA转染的PC12细胞清除ROS的能力都受到了显著损伤。结论丙炔苯丙胺可以通过激活PI3K/Akt和Erk两条激酶通路,促进Nrf2核转位发挥抗氧化效应,实现对MPP+导致PC12细胞损伤的保护作用。这种机制与丙炔苯丙胺对MAOB的抑制作用是并行的。更多还原

【Abstract】 OBJECTIVEThis part of the study was to investigate that whether deprenyl can exert neuroprotective effect against 1-methyl-4-phenylpyridinium ion (MPP+)-induced injury in PC12 cell modes of Parkinson’s disease (PD) and what the mechanism underling is.METHODSPC12 cells treated with MPP+ were used to build the cell model of PD. For investigating the neuroprotective mechanism of deprenyl against the cell damage induced by MPP+, several methods including MTT assay, lactate dehydrogenase (LDH) release analysis, reactive oxygen species (ROS) assay, nitroblue tetrazolium (NBT)/glycinate assay, and western blot were used.RESULTSThe results of MTT and LDH release analysis suggested that, the deprenyl pretreatment significantly reduced MPP+-induced PC12 cell damage in a wide range of concentration. The results of determination of ROS and NBT/glycinate assay demonstrated that deprenyl pretreatment significantly reversed the MPP+-induced enhancement of biomarkers representing oxidative stress injury. The western blotting assay for NQO1 protein suggested that, deprenyl can lead a increasing of NQO1 expression in a dose-dependent manner. As we expected, the enzymatic activity assay also showed similar result. Further, when PC12 cells were co-treated by the NQO1 inhibitor dicoumarol, the deprenyl’s cyto-protective effect was reversed as shown in LDH release analysis results.CONCLUSIONSDeprenyl can reduce the products of biomarkers representing oxidative stress injury, then exert a protective effect against MPP+-induced PC12 cell injury via up-regulating the expression of the antioxidant protein NQO1. OBJECTIVEThe objective of this part was to verify that whether deprenyl can increase the expression and enzymatic activity of anti-oxidative protein NQO1 through promoting the nuclear translocation of nuclear factor erythroid 2 related factor 2 (Nrf2) in PC12 cells against MPP+-induced cell injury.METHODSThe nuclear protein and cytoplasmic protein extraction kit was used to extract the cytoplasmic and nuclear protein from PC12 cells, respectively. Then the Nrf2 content in nuclear or cytoplasm was detected by Western blots to observe the dynamic changes among different groups. Transfection with Nrf2 SiRNA was then applied to further verify the involvement of Nrf2 nuclear translocation in deprenyl-mediated enhancement of NQO1 activity.RESULTSResults of western blots showed that, when MPP+ was added in the culture alone, both the Nrf2 level in cytoplasm and nucleus were decreased. When the PC12 cells were pretreated with deprenyl, it is found that Nrf2 in cytoplasm was decreased and the Nrf2 in nucleus was increased significantly. Further, the level of Nrf2 in cytoplasm from PC12 cells co-treated by deprenyl and MPP+ was even lower than that in cells treated by MPP+ alone. In the PC12 cells transfected withNrf2 SiRNA, the NQO1 enzymatic activity appeared significantly reduced. In addition, when Nrf2 gene was silenced, the up-regulation of NQO1 activity in deprenyl-treated PC12 cells had disappeared.CONCLUSIONS Deprenyl can increase the expression and enzymatic activity of anti-oxidative protein NQO1 through promoting the nuclear translocation of Nrf2 in PC12 cells, and then exert a cyto-protective effect against MPP+-induced cell injury. OBJECTIVEWe want to explore the upstream kinase pathway for regulating the nuclear translocation of Nrf2 in PC12 cells treated by deprenyl, and to verify that whether deprenyl’s inhibitory effect to monoamine oxidase type B (MAOB) was involved in the regulation mechanisms of Nrf2 nuclear translocation.METHODSWestern blot was used for detecting the phosphorylation level of Akt and extracellular signal-regulated kinase (Erk) in PC12 cells treated by MPP+ and /or deprenyl. In order to analyze the effect of various kinases on the regulation of Nrf2 and NQO1, inhibitors of different kinases were added into the culture. Furthermore, the MAOB and Nrf2 SiRNA were transfected into PC12 cells to analyze weather deprenyl’s inhibition to the MAOB was involved in its anti-oxidative effect mediated by nuclear translocation of Nrf2.RESULTSBased on the results of western blots in PC12 cells, we found that deprenyl treatment can induce an enhancement in the phosphorylation level of Akt with a time-dependent manner. In addition, the Nrf2 nuclear translocation and increasing expression of NQO1 mediated by deprenyl could be partially blocked by PD98059, while it was almost completely abolished by PI3K inhibitor LY294002. The results of ROS determination in PC12 cells transfect with MAOB and/or Nrf2 showed that, MAOB gene silencing can make a small amount of decline in ROS levels, but does not affect the anti-oxidative activity of deprenyl. And, regardless of MAOB gene silencing or not, the Nrf2 SiRNA transfection could hurt the ability to remove ROS significantly in PC12 cells.CONCLUSIONSDeprenyl can activate both PI3K/Akt and Erk kinase pathways, and promote nuclear translocation of Nrf2, then play an anti-oxidative effect against injury in PC12 cells treated with MPP+. And this mechanism of deprenyl is parallel with its inhibition to MAOB.更多还原