促红细胞生成素对帕金森病模型的保护作用及其机制的实验研究
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摘要
第一部分促红细胞生成素受体在1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶致C57BL/6小鼠多巴胺能神经元损伤模型不同脑区的表达变化
目的探讨促红细胞生成素受体(Erythropoietin receptor, EPOR)在1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methy1-4-phenvl-1, 2, 3, 6-tetrahvdropvridine, MPTP)致C57BL/6小鼠多巴胺能神经元损伤模型中不同脑区不同时程的表达变化。
方法采用腹腔注射MPTP制备C57BL/6小鼠PD模型。36只雄性C57BL/6小鼠,随机分为7组(n=12只):对照组、MPTP1d组、MPTP2d组、MPTP4d组、MPTP7d组、MPTP14d组、MPTP21d组。在末次注射MPTP后的相应时间将动物处死,Western blot法检测腹侧中脑、纹状体、皮质区EPOR的表达;免疫组化法检测腹侧中脑、纹状体、皮质区EPOR阳性细胞数。
结果MPTP致伤显著增加C56BL/6小鼠腹侧中脑EPOR的表达,在末次注射MPTP4天后开始升高,并且进行性增加至21天。MPTP对纹状体区与皮质区EPOR的表达均无显著性影响。
结论MPTP所致腹侧中脑区EPOR蛋白表达增加可能与PD发生发展的病理机制有关。
第二部分促红细胞生成素对1-甲基-4-苯基吡啶离子致PC12细胞损伤的抗凋亡作用及其机制研究
目的探讨促红细胞生成素(Erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(1-methy14-phenylpyridinium,MPP+)诱导PC12细胞变性损伤的保护作用及其机制。方法以MPP+损伤PC12细胞为帕金森病细胞模型,将培养的PC12细胞,分为3组:空白对照组、MPP+组、EPO+MPP+组。对照组给予无血清的DMEM培养基培养,MPP+组给予500μmol/L的MPP+处理24 h,EPO+MPP+组给予不同浓度的EPO(0.1U、0.3 U、1 U、3 U、10 U/ml)与500μmol/L的MPP+同时处理24 h。上述各组细胞加药孵育24 h后,采用四甲基偶氮唑盐(MTT)法检测细胞存活率,流式细胞术与TUNEL法检测细胞凋亡,2′,7′-dichlorofluorescin diacetate (DCF-DA)染色检测细胞内活性氧(ROS)的含量,罗丹明123 (Rhodamine123, Rh123)染色检测细胞线粒体膜电位(△Ψm),荧光法检测Caspase-3活性,Western blot法检测Bax与Bcl-2蛋白表达。
结果①500μmol/L的MPP+可以导致PC12细胞存活率下降,凋亡率增加;0.1-10 U/ml的EPO可以使PC12细胞存活率增加,凋亡率降低,并在1U/ml时达到最大保护效应;②MPP+导致ROS含量增加、△Ψm降低、Caspase-3活性增加; EPO可以抑制MPP+所致ROS、△Ψm和Caspase-3水平的变化;③MPP+组较空白对照组相比,Bax/Bcl-2比率增加;而EPO-MPP+组较MPP+组相比,Bax/Bcl-2比率降低。
结论EPO对MPP+致PC12细胞损伤具有保护作用,其机制是通过EPO抗氧化应激及抗凋亡的特性而实现的。
第三部分促红细胞生成素对MPP+致PC12细胞损伤的保护作用通路
实验一Akt/GSK-3β/Caspase-3通路在促红细胞生成素对MPP+致PC12细胞损伤保护中的作用
目的研究EPO对MPP+致PC12细胞损伤保护作用中Akt/GSK-3β/Caspase-3通路的作用。
方法以500μmol/L的MPP+损伤PC12细胞为帕金森病细胞模型,实验分组如下:空白对照组、LY294002组、LiCl组、MPP+组、MPP++EPO组、MPP++LiCl组、MPP++EPO+LY294002组。空白对照组、LY294002组、LiCl组、MPP+组、MPP++EPO组分别给予无血清DMEM培养基、10μmol/L的LY294002、20 mmol/L的LiCl、500μmol/L的MPP+、500μmol/L的MPP+ +1 U/ml的EPO处理;MPP++LiCl组先给予20 mmol/L的LiCl,1 h后给予500μmol/L的MPP+处理;MPP++EPO+LY294002组先给予10μmol/L的LY294002,1 h后给予500μmol/L的MPP+ +1 U/ml的EPO处理。孵育24 h后采用MTT法检测细胞存活率,TUNEL法检测细胞凋亡;孵育0.5 h、1 h、3 h、6 h、12 h之后采用检测Western免疫印迹法检测Akt、p-Akt、GSK-3β、p-GSK-3β蛋白表达;孵育0 h、4 h、8 h、16 h之后采用荧光法检测Caspase-3活性的变化。
结果①MPP+和/或EPO均可以诱导p-Akt表达增加;加用LY294002后p-Akt表达降低。②应用LY294002拮抗了EPO对于PC12细胞的保护作用。③MPP+和/或EPO均可以诱导p-GSK-3β表达增加;加用LY294002后p-GSK-3β表达降低。④应用LiCl拮抗了MPP+对于PC12细胞的损伤作用。⑤MPP+致PC12细胞内Caspase-3活性增加,EPO和LiCl对MPP+所致的Caspase-3活性增加均有抑制作用;应用LY294002拮抗了EPO对Caspase-3活性的抑制作用。
结论EPO对MPP+致PC12细胞损伤的保护作用是通过Akt/GSK-3b/Caspase-3通路发挥作用。.
实验二ERK通路在促红细胞生成素对MPP+致PC12细胞损伤保护中的作用
目的研究EPO对MPP+致PC12细胞损伤保护作用中ERK通路的作用。
方法以500μmol/L的MPP+损伤PC12细胞为帕金森病细胞模型,实验分组如下:空白对照组、PD98059组、MPP+组、MPP++EPO组、MPP++EPO+PD98059组。空白对照组、PD98059组、MPP+组、MPP++EPO组分别给予无血清DMEM培养基、50μmol/L的PD98059、500μmol/L的MPP+、500μmol/L的MPP+ +1 U/ml的EPO处理; MPP++EPO+ PD98059组先给予50μmol/L的PD98059 ,1 h后给予500μmol/L的MPP+ +1 U/ml的EPO处理。上述各组孵育24 h后采用四甲基偶氮唑盐(MTT)法检测细胞存活率,TUNEL法检测细胞凋亡;孵育0.5 h、1 h、3 h、6 h、12 h之后采用Western免疫印迹法检测p-ERK、ERK蛋白表达。
结果①MPP+和/或EPO均可以诱导p-ERK表达短暂增加;加用PD98059后p-ERK表达降低。②应用PD98059并不能拮抗EPO对于PC12细胞的保护作用。
结论EPO可以短暂激活ERK通路,但ERK通路并没有在EPO对MPP+致PC12细胞损伤的保护作用中发挥作用。
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 mices
Objective 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 cells
Objective 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 cells
Section 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
目的探讨促红细胞生成素受体(Erythropoietin receptor, EPOR)在1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methy1-4-phenvl-1, 2, 3, 6-tetrahvdropvridine, MPTP)致C57BL/6小鼠多巴胺能神经元损伤模型中不同脑区不同时程的表达变化。
方法采用腹腔注射MPTP制备C57BL/6小鼠PD模型。36只雄性C57BL/6小鼠,随机分为7组(n=12只):对照组、MPTP1d组、MPTP2d组、MPTP4d组、MPTP7d组、MPTP14d组、MPTP21d组。在末次注射MPTP后的相应时间将动物处死,Western blot法检测腹侧中脑、纹状体、皮质区EPOR的表达;免疫组化法检测腹侧中脑、纹状体、皮质区EPOR阳性细胞数。
结果MPTP致伤显著增加C56BL/6小鼠腹侧中脑EPOR的表达,在末次注射MPTP4天后开始升高,并且进行性增加至21天。MPTP对纹状体区与皮质区EPOR的表达均无显著性影响。
结论MPTP所致腹侧中脑区EPOR蛋白表达增加可能与PD发生发展的病理机制有关。
第二部分促红细胞生成素对1-甲基-4-苯基吡啶离子致PC12细胞损伤的抗凋亡作用及其机制研究
目的探讨促红细胞生成素(Erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(1-methy14-phenylpyridinium,MPP+)诱导PC12细胞变性损伤的保护作用及其机制。方法以MPP+损伤PC12细胞为帕金森病细胞模型,将培养的PC12细胞,分为3组:空白对照组、MPP+组、EPO+MPP+组。对照组给予无血清的DMEM培养基培养,MPP+组给予500μmol/L的MPP+处理24 h,EPO+MPP+组给予不同浓度的EPO(0.1U、0.3 U、1 U、3 U、10 U/ml)与500μmol/L的MPP+同时处理24 h。上述各组细胞加药孵育24 h后,采用四甲基偶氮唑盐(MTT)法检测细胞存活率,流式细胞术与TUNEL法检测细胞凋亡,2′,7′-dichlorofluorescin diacetate (DCF-DA)染色检测细胞内活性氧(ROS)的含量,罗丹明123 (Rhodamine123, Rh123)染色检测细胞线粒体膜电位(△Ψm),荧光法检测Caspase-3活性,Western blot法检测Bax与Bcl-2蛋白表达。
结果①500μmol/L的MPP+可以导致PC12细胞存活率下降,凋亡率增加;0.1-10 U/ml的EPO可以使PC12细胞存活率增加,凋亡率降低,并在1U/ml时达到最大保护效应;②MPP+导致ROS含量增加、△Ψm降低、Caspase-3活性增加; EPO可以抑制MPP+所致ROS、△Ψm和Caspase-3水平的变化;③MPP+组较空白对照组相比,Bax/Bcl-2比率增加;而EPO-MPP+组较MPP+组相比,Bax/Bcl-2比率降低。
结论EPO对MPP+致PC12细胞损伤具有保护作用,其机制是通过EPO抗氧化应激及抗凋亡的特性而实现的。
第三部分促红细胞生成素对MPP+致PC12细胞损伤的保护作用通路
实验一Akt/GSK-3β/Caspase-3通路在促红细胞生成素对MPP+致PC12细胞损伤保护中的作用
目的研究EPO对MPP+致PC12细胞损伤保护作用中Akt/GSK-3β/Caspase-3通路的作用。
方法以500μmol/L的MPP+损伤PC12细胞为帕金森病细胞模型,实验分组如下:空白对照组、LY294002组、LiCl组、MPP+组、MPP++EPO组、MPP++LiCl组、MPP++EPO+LY294002组。空白对照组、LY294002组、LiCl组、MPP+组、MPP++EPO组分别给予无血清DMEM培养基、10μmol/L的LY294002、20 mmol/L的LiCl、500μmol/L的MPP+、500μmol/L的MPP+ +1 U/ml的EPO处理;MPP++LiCl组先给予20 mmol/L的LiCl,1 h后给予500μmol/L的MPP+处理;MPP++EPO+LY294002组先给予10μmol/L的LY294002,1 h后给予500μmol/L的MPP+ +1 U/ml的EPO处理。孵育24 h后采用MTT法检测细胞存活率,TUNEL法检测细胞凋亡;孵育0.5 h、1 h、3 h、6 h、12 h之后采用检测Western免疫印迹法检测Akt、p-Akt、GSK-3β、p-GSK-3β蛋白表达;孵育0 h、4 h、8 h、16 h之后采用荧光法检测Caspase-3活性的变化。
结果①MPP+和/或EPO均可以诱导p-Akt表达增加;加用LY294002后p-Akt表达降低。②应用LY294002拮抗了EPO对于PC12细胞的保护作用。③MPP+和/或EPO均可以诱导p-GSK-3β表达增加;加用LY294002后p-GSK-3β表达降低。④应用LiCl拮抗了MPP+对于PC12细胞的损伤作用。⑤MPP+致PC12细胞内Caspase-3活性增加,EPO和LiCl对MPP+所致的Caspase-3活性增加均有抑制作用;应用LY294002拮抗了EPO对Caspase-3活性的抑制作用。
结论EPO对MPP+致PC12细胞损伤的保护作用是通过Akt/GSK-3b/Caspase-3通路发挥作用。.
实验二ERK通路在促红细胞生成素对MPP+致PC12细胞损伤保护中的作用
目的研究EPO对MPP+致PC12细胞损伤保护作用中ERK通路的作用。
方法以500μmol/L的MPP+损伤PC12细胞为帕金森病细胞模型,实验分组如下:空白对照组、PD98059组、MPP+组、MPP++EPO组、MPP++EPO+PD98059组。空白对照组、PD98059组、MPP+组、MPP++EPO组分别给予无血清DMEM培养基、50μmol/L的PD98059、500μmol/L的MPP+、500μmol/L的MPP+ +1 U/ml的EPO处理; MPP++EPO+ PD98059组先给予50μmol/L的PD98059 ,1 h后给予500μmol/L的MPP+ +1 U/ml的EPO处理。上述各组孵育24 h后采用四甲基偶氮唑盐(MTT)法检测细胞存活率,TUNEL法检测细胞凋亡;孵育0.5 h、1 h、3 h、6 h、12 h之后采用Western免疫印迹法检测p-ERK、ERK蛋白表达。
结果①MPP+和/或EPO均可以诱导p-ERK表达短暂增加;加用PD98059后p-ERK表达降低。②应用PD98059并不能拮抗EPO对于PC12细胞的保护作用。
结论EPO可以短暂激活ERK通路,但ERK通路并没有在EPO对MPP+致PC12细胞损伤的保护作用中发挥作用。
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 mices
Objective 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 cells
Objective 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 cells
Section 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
引文
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