Study On The Protective Effects And Mechanism Of Deprenyl Preconditioning On MPP~+-induced Injury In PC12 Cell Model Of Parkinson's Disease

PART.1 Deprenyl protect PC12 cells against injury induced by 1-Methyl-4-phenylpyridinium ion via up-regulating the expression of the antioxidant protein NAD(P)H/quinone oxidoreductase 1.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 PC 12 cell modes of Parkinson's disease (PD) and what the mechanism underling is.METHODSPC 12 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 PC 12 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 PC 12 cells were co-treated by the NQO1 inhibitor dicoumarol. the deprenyl's cyto-protective effect was reversed as shown in LDH release analvsis results.CONCLUSIONSDeprenyl can reduce the products of biomarkers representing oxidative stress injury, then exert a protective effect against MPP+-induced PC 12 cell injury via up-regulating the expression of the antioxidant protein NQO1. PART.2 Deprenyl pretreatment increased the expression and enzymatic activity of NQO1 mediated by promoting the nuclear translocation of Nrf2 in MPP+-treated PC12 cells.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 PC 12 cells against MPP+-induced cell injury.METHODSThe nuclear protein and cytoplasmic protein extraction kit was used to extract the cytoplasmic and nuclear protein from PC 12 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 PC 12 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 PC 12 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 PC 12 cells had disappeared.CONCLUSIONS Deprenyl can increase the expression and enzymatic activity of anti-oxidative protein NQ01 through promoting the nuclear translocation of Nrf2 in PC 12 cells, and then exert a cyto-protective effect against MPP+-induced cell injury. PART.3 The role of Akt and Erk signaling pathway in the anti-oxidation mechanism involved in deprenyl's protection against MPP+-induced injury in PC12 cells.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 PC 12 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 PC 12 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 PC 12 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 PC 12 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 PC 12 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 PC 12 cells treated with MPP+. And this mechanism of deprenyl is parallel with its inhibition to MAOB.