Protective Effects And Mechanisms Of Erythropoietin On Parkinsonian Model

Part I The change of expression of erythropoietin receptor in certain brain regions of 1-methy1-4-phenvl-1, 2, 3, 6-tetrahvdropvridine Parkinson disease model micesObjective To investigate the change of expression of erythropoietin receptor (EPOR) in certain brain regions on 1-methy1-4-phenvl-1, 2, 3, 6-tetrahvdropvridine (MPTP) lesioned Parkison disease model mice.Methods①Establishing MPTP lesioning of PD model mice: At the beginning of the experiment, the mice (12 per group) received an intraperitoneal (i.p.) injection of MPTP-HCl in saline at 24 h intervals for 5 days. The control group was injected with saline only. The mice were sacrificed at 1, 2, 4, 7, 14 and 21 days after the last MPTP injection.②Western blot: Western blot was carried out using total protein isolated from ventral mesencephalon, striatum and cortex, which were removed rapidly on ice from decapitated mice.③Immunohistochemistry: Immunohistochemistry experiments were carried out using tissue section prepared from ventral mesencephalon, striatum and cortex, which rapidly removed from sacrificed mice. Change of the number of EPO-R positive cells was identified using hemi-quantitive method.Results 4 day after the last MTPT treatment, the expression of EPOR and the number of EPO-R positive cells significantly increased in ventral mesencephalon compared to control group, the increased tendency continued until the 21day. While these index in striatum and cortex were not statistically different compared with those in animals that did not undergo MPTP insult.Conclusion The results showed that EPOR expression in ventral mesencephalon increased significantly after MPTP injection which strongly indicated that there were certain relationships between EPOR and the development of PD.Part II Protective effects and mechanisms of erythropoietin on 1-methy14-phenylpyridinium -induced neurodegeneration in PC12 cellsObjective The neuroprotective effect of erythropoietin (EPO) against 1-methyl-4- phenylpyridinium (MPP+)-induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated.Methods PC12 cells impaired by MPP+ were used as the cell model of Parkinson's disease. The cultured cells were divide into 3 groups: control group was treated with DMEM medium, MPP+ group was treated with 500μmol/L MPP+, MPP+ and EPO group was co-incubated with 500μmol/L MPP+ and different concentrations (0, 0.1, 0.3, 1, 3, or 10 U/mL) of EPO. After incubation for 24 h, methyl thiazolyl tetrazolium (MTT) was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, flow cytometry and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay were used to analyze the apoptosis ratio of PC12 cells. The expression of Bcl-2 and Bax in PC12 cells were examined by Western blot, and the reactive oxygen species (ROS), the mitochondrial transmembrane potential and the activity of Caspase-3 in each group were detected by spectrofluorometer.Results①Treatment of PC12 cells with MPP+ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, EPO had significantly protective effect on the neurodegeneration and had the maximum protective effect at 1 U/ml.②MPP+ induced the formation of ROS, the disruption of mitochondrial transmembrane potential and the activation of Caspase-3. In contrast, EPO significantly reversed these responses.③It was also shown that MPP+ significantly induced the upregulation of Bax/Bcl-2 ratio, while EPO significantly downregulated the ratio.Conclusion EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson's disease, and the inhibitive effect of EPO on the MPP+-induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity.Part III The study of signaling pathway in the protective effect of EPO on MPP+ induced neurotoxicity in PC12 cellsSection I The role of Akt/GSK-3β/Caspase-3 signaling pathway in the protective effect of EPO on MPP+ induced neurotoxicity in PC12 cells Objective To investigate the role of Akt/GSK-3β/Caspase-3 signaling pathway in the protective effect of EPO on MPP+ induced neurotoxicity in PC12 cells.Method PC12 cells impaired by MPP+ were used as the cell model of Parkinson's disease. The cultured cells were divide into such groups: control group received the administration of DMEM medium; LY294002 group, LiCl group and MPP+ group were identical to the control group except that LY294002 (10μmol/L), LiCl (20 mmol/L) or MPP+ (500μmol/L) was administered instead of DMEM medium, respectively; MPP++EPO group was treated with MPP+(500μmol/L) and EPO(1U/ml); MPP++LiCl group received LiCl (20 mmol/L) 1 h before treatment with MPP+(500μmol/L); MPP++EPO+LY294002 group was identical to the MPP++EPO group except that LY294002 was given 1 h before MPP+ and EPO. After incubation for 24 h, methyl thiazolyl tetrazolium (MTT) was used to assay the viability of the PC12 cells and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay were used to analyze the apoptosis ratio of PC12 cells. After incubation for 0.5 h, 1 h, 3 h, 6 h, 12 h, western blot was used to detect the expression level of Akt, p-Akt, GSK-3β, p-GSK-3β. After incubation for 0 h, 4 h, 8 h, 16 h, the activity of Caspase-3 was detected by spectrofluorometer.Results①Enhanced levels of p-Akt were detected EPO was added to the culture and there was a significant increase in the phosphorylation of Akt in MPP+-treated cells by EPO. LY294002 thoroughly abolished EPO-induced phosphorylation of Akt.②The effect of rescuing the cell from death induced by EPO was lost with addition of LY 294002, a specific inhibitor of PI3K.③Phosphorylation of GSK-3βwas enhanced following EPO-treated and there was a significant increase in the phosphorylation of GSK-3βin MPP+-treated cells by EPO. LY294002 pretreatment abolished phosphorylation of GSK-3βby EPO.④Inhibition of GSK-3βby LiCl promoted viability and reduced apoptosis in PC12 cells subjected to MPP+.⑤MPP+ induced a time dependent increase in caspase-3-like proteinase activities. EPO and LiCl significantly decreased caspase 3-like activities induced by MPP+ toxicity. The addition of the PI3K inhibitor, LY294002 in PC12 cells reversed the effect of EPO on Caspase-3 activation.Conclusion Thus, these findings indicate that EPO protects against apoptosis in PC12 cells exposed to MPP+, through the Akt/GSK-3b/ Caspase-3 signaling pathway in this model system. EPO; MPP~+ ; PC12 cell; ERK; apoptosis...