Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of B...Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.展开更多
BACKGROUND： Cyclophilin A can protect neurons against oxidative stress. OBJECTIVE： To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochro-mocytoma （PC12） cells treated with beta...BACKGROUND： Cyclophilin A can protect neurons against oxidative stress. OBJECTIVE： To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochro-mocytoma （PC12） cells treated with beta-amyloid fragment 25-35 （Aβ25-35）, and to verify the protection pathway of cyclophilin A. DESIGN, TIME AND SETTING： The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007. MATERIALS： PC12 cells were cultured at the Cell Center of Peking Union Medical College. Aβ25-35 （Sigma, USA）, antibodies of Bcl-2 and Bax （Wuhan Boster, China）, and recombinant human cyclophilin A （Biomol, USA） were used in this study. METHODS： PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μmol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final con-centration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES： After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells. RESULTS： Bcl-2 expression decreased, whereas Bax expression increased in PC12 cells treated with Aβ25-35 （t = 2.277, 5.957, P 〈 0.05）. However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased （t = 4.497, 2.531, P 〈 0.05）. The survival rate of PC12 cells significantly decreased and the apoptosis rate increased （t=8.509, 22.886, P 〈 0.05） following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis （t = 4.895, 10.042, P 〈 0.05）. CONCLUSION： Cyclophilin A can increase Bcl-2 expression and decrease Bax expression in PC12 cells treated with Aβ25-35, which indicates that cyclophilin A has a protective effect on Aβ25-35-induced injury to PC12 cells.展开更多
目的:探索缺氧预适应小鼠脑匀浆提取液(Brain tissue extract of hypoxia-preconditioned mice,BHP)对NGF诱导分化的PC12细胞缺氧耐受性的影响及保护性作用的适合浓度。方法:复制小鼠急性重复缺氧模型,制备BHP,用考马斯亮蓝法定量蛋白...目的:探索缺氧预适应小鼠脑匀浆提取液(Brain tissue extract of hypoxia-preconditioned mice,BHP)对NGF诱导分化的PC12细胞缺氧耐受性的影响及保护性作用的适合浓度。方法:复制小鼠急性重复缺氧模型,制备BHP,用考马斯亮蓝法定量蛋白。在分化PC12细胞中加入BHP使其蛋白终浓度分别为1.0、10.0、100.0μg/ml(BHP组)。以等蛋白终浓度正常小鼠脑匀浆提取液(Brain tissue extract of normal mice,BN)作为对照组(BN组)。同时设置只加PBS的溶剂对照组(PBS组)。缺氧(2%o_2)培养24、48、72h后,采用回唑盐(MTT)比色法检测各组细胞活力(OD_(570nm)值)和乳酸脱氢酶(LDH)透出率、缺氧72h晚期凋亡率。结果:缺氧24h时,蛋白终浓度为10.0、100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组和缺氧PBS组,其LDH透出率显著低于同浓度BN组和缺氧PBS组。随缺氧时间延长,其保护作用逐渐减弱。至缺氧72h时,仅蛋白终浓度为100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组,其LDH透出率、晚期凋亡率均显著低于缺氧PBS组。结论:BHP对分化PC12细胞缺氧耐受性的增强作用有浓度依赖性和时间依赖性,蛋白终浓度为100.0μg/ml的BHP组保护作用稳定、持久。展开更多
Oxidative stress has an important role in the development of Alzheimer＇s disease （AD）. Beta amyloid protein 25-35 （Aβ25-35） can generate oxygen free radicals, and MCI-186 （3-methyl-l-phenyl-2-pyrazolin-5-one, e...Oxidative stress has an important role in the development of Alzheimer＇s disease （AD）. Beta amyloid protein 25-35 （Aβ25-35） can generate oxygen free radicals, and MCI-186 （3-methyl-l-phenyl-2-pyrazolin-5-one, edaravone） can specifically eliminate hydroxyl radicals. The present study introduced Aβ25-35 into PC12 cells to establish a cell model of AD, and investigated the neuroprotective effects of MCI-186 on AD. Results showed that MCI-186 had a positive effect on the prevention and treatment of AD by inhibiting protein oxidative products, advanced glycation end products, lipid oxidative end products and DNA oxidative damage in PC12 cells induced by Aβ25-35.展开更多
Neuronal growth inhibitory factor (GIF), named Metallothioneins-III (MT-III), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombi...Neuronal growth inhibitory factor (GIF), named Metallothioneins-III (MT-III), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochro-mocytoma PC12 by the MTT (Thiazolyl blue) reduction as-say. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for de-tecting neuronal growth inhibitory activity of GIF. Recom-binant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhib-iting PC12 may be that PC12 have some properties of cho-linergic neuron. It must play an important role in discover-ing the mechanism of GIF’s neuronal growth inhibitory ac-tivity.展开更多
Ferulic acid (FA) is a ubiquitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have revealed that FA shows neuroprotective effect and significant antidepressant- lik...Ferulic acid (FA) is a ubiquitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have revealed that FA shows neuroprotective effect and significant antidepressant- like effect. The aim of this study was to investigate its potential neurogenesis-enhancing effect and its role in repair following stress-induced neuronal damage. MTT assay was performed to measure the effect of SF on the growth of rat pheochromocytoma (PC12) cells;morphological and immunocytochemical meth- ods were used for assessing its differentiation-induc- ing action. Chronic mild stress (CMS) tests were per- formed to establish rat model of depression. The histopathology of animal brains was studied to ana- lyze CMS-induced morphological changes and the effect of SF on the repair of CMS-induced brain in- jury. The expressions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the proliferation of neural stem cell/neural progenitor cells were assessed in the hippocampi of chronic mild stress (CMS)-induced depression-like model rats by immunohistochemistry and bromodeoxyuridine (BrdU)- incorporation assays, respectively. Our in vitro tests showed that SF promoted the proliferation of PC12 cells in the concentration range of 5 - 320 μM, and induced PC12 cells to differentiate to more mature cells with the morphological characteristics and mo- lecular marker of neuronal-like cells. In vivo tests showed that SF up-regulated the expressions of NGF and BDNF, and induced the proliferation of neural stem cell/neural progenitor cells in the hippocampi of CMS-induced depression-like model rats. This study provides evidences that SF shows neurogenesis-en- hancing effect, and its antidepressant-like effect of SF may be related directly and closely to its above-men- tioned effect.展开更多
基金Project (No. 2005C30059) supported by the Science and TechnologyProgram of Zhejiang Province, China
文摘Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.
文摘BACKGROUND： Cyclophilin A can protect neurons against oxidative stress. OBJECTIVE： To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochro-mocytoma （PC12） cells treated with beta-amyloid fragment 25-35 （Aβ25-35）, and to verify the protection pathway of cyclophilin A. DESIGN, TIME AND SETTING： The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007. MATERIALS： PC12 cells were cultured at the Cell Center of Peking Union Medical College. Aβ25-35 （Sigma, USA）, antibodies of Bcl-2 and Bax （Wuhan Boster, China）, and recombinant human cyclophilin A （Biomol, USA） were used in this study. METHODS： PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μmol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final con-centration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES： After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells. RESULTS： Bcl-2 expression decreased, whereas Bax expression increased in PC12 cells treated with Aβ25-35 （t = 2.277, 5.957, P 〈 0.05）. However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased （t = 4.497, 2.531, P 〈 0.05）. The survival rate of PC12 cells significantly decreased and the apoptosis rate increased （t=8.509, 22.886, P 〈 0.05） following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis （t = 4.895, 10.042, P 〈 0.05）. CONCLUSION： Cyclophilin A can increase Bcl-2 expression and decrease Bax expression in PC12 cells treated with Aβ25-35, which indicates that cyclophilin A has a protective effect on Aβ25-35-induced injury to PC12 cells.
文摘目的:探索缺氧预适应小鼠脑匀浆提取液(Brain tissue extract of hypoxia-preconditioned mice,BHP)对NGF诱导分化的PC12细胞缺氧耐受性的影响及保护性作用的适合浓度。方法:复制小鼠急性重复缺氧模型,制备BHP,用考马斯亮蓝法定量蛋白。在分化PC12细胞中加入BHP使其蛋白终浓度分别为1.0、10.0、100.0μg/ml(BHP组)。以等蛋白终浓度正常小鼠脑匀浆提取液(Brain tissue extract of normal mice,BN)作为对照组(BN组)。同时设置只加PBS的溶剂对照组(PBS组)。缺氧(2%o_2)培养24、48、72h后,采用回唑盐(MTT)比色法检测各组细胞活力(OD_(570nm)值)和乳酸脱氢酶(LDH)透出率、缺氧72h晚期凋亡率。结果:缺氧24h时,蛋白终浓度为10.0、100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组和缺氧PBS组,其LDH透出率显著低于同浓度BN组和缺氧PBS组。随缺氧时间延长,其保护作用逐渐减弱。至缺氧72h时,仅蛋白终浓度为100.0μg/ml的BHP组OD_(570nm)值显著高于同浓度BN组,其LDH透出率、晚期凋亡率均显著低于缺氧PBS组。结论:BHP对分化PC12细胞缺氧耐受性的增强作用有浓度依赖性和时间依赖性,蛋白终浓度为100.0μg/ml的BHP组保护作用稳定、持久。
基金the Talent Introduction Project of Affili-ated Hospital of Jiangsu University,No.jdfyRC 2008003
文摘Oxidative stress has an important role in the development of Alzheimer＇s disease （AD）. Beta amyloid protein 25-35 （Aβ25-35） can generate oxygen free radicals, and MCI-186 （3-methyl-l-phenyl-2-pyrazolin-5-one, edaravone） can specifically eliminate hydroxyl radicals. The present study introduced Aβ25-35 into PC12 cells to establish a cell model of AD, and investigated the neuroprotective effects of MCI-186 on AD. Results showed that MCI-186 had a positive effect on the prevention and treatment of AD by inhibiting protein oxidative products, advanced glycation end products, lipid oxidative end products and DNA oxidative damage in PC12 cells induced by Aβ25-35.
基金This work was supported by the National Ninth Five-Year Plan High Technology Projects (Grant No. 96-C02-01-09).
文摘Neuronal growth inhibitory factor (GIF), named Metallothioneins-III (MT-III), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochro-mocytoma PC12 by the MTT (Thiazolyl blue) reduction as-say. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for de-tecting neuronal growth inhibitory activity of GIF. Recom-binant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhib-iting PC12 may be that PC12 have some properties of cho-linergic neuron. It must play an important role in discover-ing the mechanism of GIF’s neuronal growth inhibitory ac-tivity.
文摘Ferulic acid (FA) is a ubiquitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have revealed that FA shows neuroprotective effect and significant antidepressant- like effect. The aim of this study was to investigate its potential neurogenesis-enhancing effect and its role in repair following stress-induced neuronal damage. MTT assay was performed to measure the effect of SF on the growth of rat pheochromocytoma (PC12) cells;morphological and immunocytochemical meth- ods were used for assessing its differentiation-induc- ing action. Chronic mild stress (CMS) tests were per- formed to establish rat model of depression. The histopathology of animal brains was studied to ana- lyze CMS-induced morphological changes and the effect of SF on the repair of CMS-induced brain in- jury. The expressions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the proliferation of neural stem cell/neural progenitor cells were assessed in the hippocampi of chronic mild stress (CMS)-induced depression-like model rats by immunohistochemistry and bromodeoxyuridine (BrdU)- incorporation assays, respectively. Our in vitro tests showed that SF promoted the proliferation of PC12 cells in the concentration range of 5 - 320 μM, and induced PC12 cells to differentiate to more mature cells with the morphological characteristics and mo- lecular marker of neuronal-like cells. In vivo tests showed that SF up-regulated the expressions of NGF and BDNF, and induced the proliferation of neural stem cell/neural progenitor cells in the hippocampi of CMS-induced depression-like model rats. This study provides evidences that SF shows neurogenesis-en- hancing effect, and its antidepressant-like effect of SF may be related directly and closely to its above-men- tioned effect.
