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
Objective To examine the role of Cd-induced reactive oxygen species(ROS) generation in the apoptosis of neuronal cells. Methods Neuronal cells(primary rat cerebral cortical neurons and PC12 cells) were incubated w...Objective To examine the role of Cd-induced reactive oxygen species(ROS) generation in the apoptosis of neuronal cells. Methods Neuronal cells(primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin(Rap) or N-acetyl-L-cysteine(NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B(Akt)/mammalian target of rapamycin(m TOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase(p70 S6K), and eukaryotic initiation factor 4E binding protein 1(4E-BP1). Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein(Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase(PARP), and nuclear translocation of apoptosis-inducing factor(AIF) and endonuclease G(Endo G). Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.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
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
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.
基金supported by the National Natural Science Foundation of China(No.31101866 and 31302058)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD),China Postdoctoral Science Foundation funded project(2015M581874)Jiangsu Planned Projects for Postdoctoral Research Funds(1501072A)
文摘Objective To examine the role of Cd-induced reactive oxygen species(ROS) generation in the apoptosis of neuronal cells. Methods Neuronal cells(primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin(Rap) or N-acetyl-L-cysteine(NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B(Akt)/mammalian target of rapamycin(m TOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase(p70 S6K), and eukaryotic initiation factor 4E binding protein 1(4E-BP1). Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein(Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase(PARP), and nuclear translocation of apoptosis-inducing factor(AIF) and endonuclease G(Endo G). Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.
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.
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.
基金国家自然科学基金项目(81603537,81473668)四川省科技厅国际合作与交流项目(2017HH0004)+3 种基金国家留学基金委项目(201908510279)四川省教育厅重点项目(17ZA0163)成都中医药大学青年学者人才项目(QNXZ2019043)the 2019 Internationalization Incentive Fund of University of Otago(IIFUO2019)。