Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equi...Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.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
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
基金the National Natural Science Foundation of China, No. 30672136
文摘Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.
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.
