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cyclophilin a affects Bcl-2 and Bax expression following beta-amyloid fragment 25-35-induced injury to PC12 cells

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Neural Regeneration Research 2008年第6期3卷 585-588页

作者： Li Cheng Chaodong Zhang Postgraduate of China Medical University Shenyang 110001 Liaoning Province China Department of Clinical Internal Medicine First Clinical College China Medical University Shenyang 110001 Liaoning Province China

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 in

关键词： cyclophilin a pheochromocytoma (PC12) cells β-amyloid fragment 25-35 Bcl-2 Bax

Protective effect of cyclophilin a against Alzheimer＇s amyloid beta-peptide （25-35）-induced oxidative stress in PC12 cells

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Chinese Medical Journal 2009年第6期122卷 716-724页

作者： GE Yu-song TENG Wei-yu ZHANG Chao-dong Department of Neurology First Affiliated Hospital China Medical University Shenyang Liaoning 110001 China

Background β-amyloid peptide （Aβ） is considered responsible for the pathogenesis of Alzheimer＇s disease （AD）. Possible mechanisms underlying Aβ-induced neuronal cytotoxicity include excessive production of reactive oxidative species （ROS） and apoptosis. cyclophilin a （CypA）, exhibits antioxidant properties and protects neurons against oxidative stress induced injury. This study was conducted to demonstrate whether CyPA added to cultured PC12 cells could alleviate Aβ-induced oxidative stress and protect them from apoptosis.Methods PC12 cells were pre-incubated for 30 minutes with recombinant human cyclophilin a （rhCyPA） in 0.1 nmol/L, 1.0 nmol/L, 10 nmol/L and 100 nmol/L and then incubated with 10 μmol/L Aβ25-35. In every group, cell viability, apoptotic morphology, apoptotic rate, intracellular ROS accumulation, the activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） of PC12 cells and mitochondrial transmembrane potential were detected. Subsequently, the expression of the active form of caspase-3 was determined by Western blotting.Results It was shown that cultures treated with 1.0 nmol/L, 1U nmol/L or 100 nmol/L rnL, rhCyPA＋Aβ25-35 had significantly higher cell viability and a lower rate of apoptosis compared with the cultures exposed only to Aβ25-35. In addition, rhCyPA attenuated Aβ25-35-induced overproduction of intracellular ROS and Aβ25.35-induced a decrease in activity of the key antioxidant enzymes SOD and GSH-Px. Furthermore, rhCyPA also attenuated Aβ25.35-induced mitochondrial dysfunction and the activation of caspase-3.Conclusion CyPA may act as an ROS scavenger, and prevent Aβ25-35-induced neurotoxicity through attenuating oxidative stress induced by Aβ25-35.

关键词： Alzheimer's disease cyclophilin a β-amyloid oxidative stress apoptosis

cyclophilin a Signaling Induces Pericyte-associated Blood-Brain Barrier Disruption After Subarachnoid Hemorrhage

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Cyclophilin A Signaling Induces Pericyte-associated Blood-Br...

中国生理学会基质生物学专业委员会第二次全国基质生物学学术会议

作者： Pengyu Pan Hengli Zhao Xuan Zhang Qiang Li Jie Qu Shilun Zuo Fan Yang John H.Zhang Haiyang He Xin Liu Hua Feng 陈渝杰 Department of Neurosurgery Southwest HospitalThird Military Medical University Department of Pathophysiology and High Altitude Pathology College of High Altitude Military MedicineThird Military Medical University Neuroscience Research Center Loma Linda University Institute of Immunology Third Military Medical University 第三军医大学

Within vascular neural network,blood-brain barrier damage contributes to vasogenic brain edema,which is one of the most common pathophysiological changes after SAH and has become a focus of interest in the development of novel therapeutic strategies for SAH ***,the potential roles of pericytes in maintaining the integrity of the blood-brain barrier following SAH and the exact mechanisms of this process,which would be helpful for the development of therapeutic strategies,remain *** the present study,we sought to provide evidence on the potential role of pericytes in blood-brain barrier disruption and the possible involvement and mechanism of CypA signaling in both cultured primary pericytes and SAH *** hundred and ninety-four(394) adult male C57 B6 J mice weighing 22 to 30 g,30 CypA knockout(CypA-/-) mice,and 30 male neonatal C57 B6 J mice were used to investigate the time course of CypA expression in pericytes after SAH,the intrinsic function and mechanism of CypA in the pericytes,and whether the known receptor CD147 mediates the effects of *** data demonstrated that both intracellular CypA and CypA secretion increased after SAH and could activate its receptor CD147 and the downstream NF-κB inflammatory pathway to induce MMP-9 expression and proteolytic functions for the degradation of endothelium tight junction proteins and basal *** served as an autocrine or a paracrine ligand for its receptor,*** CypA could be endocytosed by pericytes,the specific endocytosis inhibitor chlorpromazine did not have an effect on MMP-9 activation following blood-brain barrier ***,the specific knockdown of CD 147 could reverse the harmful effects of CypA expression in the pericytes on the blood-brain barrier integrity after *** study demonstrated for the first time that CypA is elevated in the pericytes of the brain after SAH,and extraneous recombinant CypA damages the blood-brain barrier and diminishes the ne

关键词： Subarachnoid hemorrhage pericyte cyclophilin a Blood-Brain Barrier endocytosis CD147

cyclophilin a结合Caveolin-1降低大鼠血管平滑肌细胞胆固醇蓄积

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Cyclophilin A结合Caveolin-1降低大鼠血管平滑肌细胞胆固醇蓄积

作者：郭琰南华大学

学位级别：硕士

目的:通过Ox-LDL诱导的血管平滑肌细胞荷脂模型,观察胆固醇转运复合物中关键蛋白cyclophilin a和caveolin-1在细胞荷脂过程中的表达变化及其与细胞内胆固醇蓄积的关系,进而分析这两种蛋白在血管平滑肌源性荷脂细胞形成过程中的作用。方... 详细信息

目的:通过Ox-LDL诱导的血管平滑肌细胞荷脂模型,观察胆固醇转运复合物中关键蛋白cyclophilin a和caveolin-1在细胞荷脂过程中的表达变化及其与细胞内胆固醇蓄积的关系,进而分析这两种蛋白在血管平滑肌源性荷脂细胞形成过程中的作用。方法:实验采用75mg/L Ox-LDL与血管平滑肌细胞共同孵育不同的时间(0,24,48,72h),建立平滑肌细胞荷脂模型;Western-blot、免疫荧光法分别定量,定位检测cyclophihn A和caveolin-1蛋白的表达改变;油红O染色观察细胞内脂滴的形成情况;高效液相色谱法检测细胞内外胆固醇含量变化,免疫共沉淀检测cyclophilin a和caveolin-1相互作用。结果:75mg/L Ox-LDL与血管平滑肌细胞共同孵育24,48,72小时后,与未处理组比较,cyclophilin a的蛋白表达逐渐减弱,同时,胆固醇转运复合物中另一关键蛋白caveolin-1的表达也逐渐降低,差异均有显著性(Pcyclophilin a与caveolin-1在胞膜、胞浆中的表达,且随着处理时间的延长,作用更明显。油红O染色显示在Ox-LDL处理细胞48小时后,细胞内有大量脂滴的形成,高效液相检测分析细胞内总胆固醇含量不断增加,细胞外胆固醇总含量随着Ox-LDL处理24、48、72小时依次下降。并且cyclosporin A抑制cyclophilin a活性后能增加细胞内总胆固醇量和降低细胞外胆固醇总量。免疫共沉淀结果显示cyclophilin a和caveolin-1在大鼠血管平滑肌细胞中能够结合,而cyclosporin A能降低cyclophilin a与caveolin-1的结合能力。结论: 1. Ox-LDL可能通过下调cyclophilin a与caveolin-1的表达促进血管平滑肌细胞内胆固醇蓄积。 2.抑制cyclophilin a活性可降低cyclophilin a与caveolin-1的相互作用,并促进细胞内的胆固醇蓄积。 3. cyclophilin a与caveolin-1的相互作用,可能是调节血管平滑肌细胞内胆固醇稳定的重要机制。

关键词： cyclophilin a 血管平滑肌细胞胆固醇 caveolin-1 cyclosporin A

cyclophilin a Inhibits Cholesterol Accumulation Via the Caveolin-1 Bonding-dependent Pathway in Vascular Smooth Muscle Cells

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Cyclophilin A Inhibits Cholesterol Accumulation Via the Cave...

第八届海峡两岸心血管科学研讨会

作者： Qin-hui Tuo~1,Yan Guo~1,Hong-yan Ling~1,Zi-fen Guo~1,Rui Yi~1,Duan-fang Liao~(2*) (1.China Institute of Pharmacy and Pharmacology,University of South China,Hengyang,China 2.Department of Traditional Chinese Diagnotics,Hunan University of Chinese Medicine,Changsha,China)

＜正＞Background Epidemiological studies have shown the intracellular decrease of cholesterol or amountreduction of foam cells could reverse *** A and caveolin - 1 as the key proteins participate in cholesteorl efflux in foam *** the function of these proteins in vascular smooth cells （VSMCs） are still not *** To observe the protein expression and function of cyclophilin a and caveolin - 1 in lipid - loaded VSMCs induced by oxidized low density lipoprotein and to investigate their effect on cellular cholesterol *** VSMCs were treated with 75 mg/LOx - LDL for different time （0,24,48,72 h）.Western - blot and immunofiuoerscence were used to detect protein expression of cyclophilin a and Caveolin - *** Red 0 dye experiment was used to show the lipid droplets in lipid - loaded *** Performance Liquid Chromatography（HPLC） analysis was performed to determine the content of cellular total cholesterol and cholesterol *** A was used to inhibit the activity of cyclopilin A. Co - immunoprecipitation（IP） was used to identify the combination between cyclophilin a and Caveolin - 1, and to observe the effect of the cyclosporin A to the combination of cyclopilin A and caveolin - *** Western - blot analysis showed that the protein expression of both cyclophilin a and Caveolin - 1 were significantly decreased in a timedependent *** showed the same trend of cyclophilin a and caveolin - *** Red 0 dye showed that there were a lot of lipid droplets accumulated in VSMCs with Ox -LDL treatment after 48 *** analysis showed that the contents of intracellular total cholesterol and cholesterol ester were increased *** is more,HPLC showed that the content of intracellular total cholesterol was increased when the VSMCs were treated with Cyclosporin *** A can combine with caveolin - 1 and the combination reduced after VSMCs being treated with cyclosporin *** the pr

关键词： cyclophilin a vascular smooth muscle cells cholesterol caveolin-1 CyclosporinA

cyclophilin a通过与Caveolin-1协同作用抑制RAW264.7细胞胆固醇蓄积

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Cyclophilin A通过与Caveolin-1协同作用抑制RAW264.7细胞胆固醇...

作者：易睿南华大学

学位级别：硕士

目的:构建鼠源性cyclophilin a和Caveolin-1基因重组质粒,瞬时转染RAW264.7细胞,建立稳定表达细胞株,然后通过ox-LDL诱导细胞荷脂,观察胆固醇转运复合物中关键蛋白cyclophilin a对Caveolin-1介导RAW264.7细胞胆固醇含量的影响。方... 详细信息

目的:构建鼠源性cyclophilin a和Caveolin-1基因重组质粒,瞬时转染RAW264.7细胞,建立稳定表达细胞株,然后通过ox-LDL诱导细胞荷脂,观察胆固醇转运复合物中关键蛋白cyclophilin a对Caveolin-1介导RAW264.7细胞胆固醇含量的影响。方法:从鼠源性RAW264.7细胞中提取总RNA,设计特异性引物,通过RT-PCR法扩增cyclophilin a和Caveolin-1基因编码区序列（CDS）片段,将其插入到质粒中,经双酶切和测序鉴定,确定重组质粒是否构建成功;将构建成功的重组质粒瞬时转染RAW264.7细胞,然后采用100mg/L ox-LDL与RAW264.7细胞共同孵育48h,建立RAW264.7细胞荷脂模型,酶荧光法观察细胞内胆固醇酯比率变化,油红O染色观察细胞内脂滴的形成情况,免疫共沉淀检测转染细胞cyclophilin a和Caveolin-1的相互作用,免疫荧光法定位检测转染细胞cyclophilin a和Caveolin-1蛋白的表达改变。结果:以重组载体为模板扩增得到的片段大小与已知cyclophilin a和Caveolin-1基因大小相同,酶切也得到目的基因片段,测序结果也证实cyclophilin a和Caveolin-1基因片段插入序列和方向正确;瞬时转染的RAW264.7细胞,与正常组和空转组比较,cyclophilin a、Caveolin-1的基因和蛋白表达均增强,有显著性差异（P＜0.05）,尤其以转染48h明显;将100mg/L ox-LDL与瞬时转染Caveolin-1组和共转染cyclophilin a和Caveolin-1组的RAW264.7细胞共同孵育48h后,酶荧光定量检测分析两组细胞内胆固醇酯占总胆固醇含量比率较未处理组和空转组均减少,尤以后者减少更为明显;油红O染色显示细胞内脂滴较未处理组和空转组减轻,尤以共转染cyclophilin a和Caveolin-1组减少更为明显;免疫共沉淀结果也说明Caveolin-1转染和cyclophilin a和Caveolin-1共转染的RAW264.7细胞,cyclophilin a和Caveolin-1的相互作用增强;间接免疫荧光定位检测发现转染Caveolin-1和共转染cyclophilin a和Caveolin-1的RAW264.7细胞中cyclophilin a和Caveolin-1在胞膜、胞浆中的表达均增强,以后者为甚。结论: 1、将含cyclophilin a和Caveolin-1质粒共同导入RAW264.7细胞可增加cyclophilin a和Caveolin-1的结合。 2、将含Caveolin-1质粒导入RAW264.7细胞可抑制ox-LDL诱导的胆固醇蓄积。 3、将含cyclophilin a和Caveolin-1质粒共同导入RAW264.7细胞可增强抑制ox-LDL诱导的胆固醇蓄积作用。

关键词： cyclophilin a Caveolin-1 真核表达载体巨噬细胞胆固醇

Role of cyclophilin a in Cellular Cholesterol Accumulation in Macrophages Induced by Oxidized Low Density Lipoprotein

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Role of Cyclophilin A in Cellular Cholesterol Accumulation i...

第十五届国际药理学大会

作者： TU Jian YAN Peng -ke ZHU Bing -yang Liao Duanfang.Division of Pharmacoproteomics Institute of Pharmacy and Pharmacology Nanhua University Hengyang 421001 China. Institute of Pharmacy and Pharmacology Nanhua University Hengyang Hunan 421001 China.Institute of Pharmarcy and Pharmacology Nanhua University Hengyang 421001 China.

＜正＞ Aim To study the roles of cyclophilin a on cellular cholesterol accumulation in macrophages derived from RAW264.7 monocyte induced by ox - LDL. Methods RAW264.7 - derived macrophages were pretreated with 75mg/L probucol for 30 min and followed by 75mg/L ox - LDL treatment for 48 hours. Western - blot and immunofluorescence were used to detect the expression of cyclophilin a. Oil Red O staining. HPLC and liquid scintillator accounting were performed to determine the cellular lipid droplets, total cholesterol （TC）. free cholesterol （FC） and cholesterol ester （CE）. Results Ox - LDL treatment increased cellular levels of TC. FC. and CE. and also increased cellular accumulation of lipid droplets, but decreased cyclophilin a expression by54.5±6.3%（p＜0.05）. Probucol pretreatmentsignificantly decreased the cellular levels of TC and CE companied by an obvious abatement of lipid droplets. Furthermore, probucol promoted cyclophilin a expression by81.3±3.6 %（p＜0.05）. Conclusion Probucol deceases cellular cholesterol accumulation induced by ox - LDL. which might be due to its up -regulation on cyclophilin a expression.

关键词： Probucol cyclophilin a lipid-loaded cells ox-LDL.

The roles of TRIM5α and cyclophilin a in host innate immune response to HIV-1

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The roles of TRIM5α and Cyclophilin A in host innate immune...

新发传染病研究热点研讨会

作者： Yi-Qun Kuang Yong-Tang Zheng Guangdong Provincial Institute of Public Health Institute of AIDS Control and Prevention Guangdong Center for Disease Control and Prevention Key Laboratory of Animal Models and Human Diseases Mechanisms of the Chinese Academy of Sciences & Yunnan Province Kunming Institute of Zoology Chinese Academy of Sciences

During long-term evolutionary history, virus and host interaction has driven the first line barrier, innate immunity, to invading pathogens. Innate immune factor TRIM5α, along with host peptidyl-prolyl cis-trans isomerase cyclophilin a, are two key players in the interaction between HIV-1 and host. Interestingly, CypA is retrotransposed into a critical host gene, TRIM5, locus via LINE-1 element in some primate species, including New World Monkeys and Old World Monkeys. This review aims to comprehensively discuss the sensing and immune activation procedures of TRIM5α innate signaling pathway through cyclophilin a. It will then present the production of TRIMCyp chimeric gene and the different fusion patterns in primates. Finally, it will summarize the distinct restriction activity of TRIMCyp from different primates, and explain the current understanding on the innate immune mechanisms involved in the early phase of the viral life cycle to HIV-1 replication.

关键词： TRIM5 cyclophilin a innate immune HIV-1

Proteomics identification of heat shock protein 27 and cyclophilin a participate in the pathogenesis of COPD

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Proteomics identification of heat shock protein 27 and cyclo...

中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： Rui-Cheng HU,Qing OUYANG,Ai-Guo DAI,Shuang-Xiang TAN,Zhi-Qiang XIAO,Cen-E TANG 1 Department of Respiratory Medicine,Hunan Institute of Gerontology,Hunan Province Geriatric Hospital,Changsha 410016, China 2 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health,Xiangya Hospital,Central South University,Changsha 410016,China

＜正＞Background and objective:COPD is a public health problem worldwide with complex pathogenesis, and has not been well *** current study aims to acquire new clues for the pathogenesis of COPD by use of proteomics ***:Lung tissue specimens were harvested from never-smokers,non-COPD smokers and COPD smokers,and the proteins differentially expressed between the 3 group subjects were revealed by two dimensional electrophoresis and sequence identified by matrix assisted laser desorption/ionization time of flight mass spectrometry analysis combined with bioinformation *** interested differentially expressed proteins identified by proteomics technology were validated by immunohistochemistory and ***: Forty seven protein spots were revealed to be differentially expressed between groups with intensity variation more than 1.5 times from two dimensional electrophoresis maps,and 24 proteins were identified through mass spectrometry analysis combined with database *** expressed proteins were functionally mainly involved in basic metabolism,oxidoreduction,coagulation/fibrinolysis,protein degradation,signal transduction, inflammation,cell growth/*** the 24 identified proteins,22 proteins were up-regulated in the lung of smokers compared with *** results of proteomics,immunohistochemical staining and Western-blotting accordant demonstrated that the expression of heat shock protein 27 and cyclophilin a in lung was increased in non-COPD smokers and further up-regulated in COPD smokers when compared with never-smokers. Conclusion:Heat shock protein 27 and cyclophilin a participate in the pathogenesis of COPD,and their expression in lung is induced by smoking.

关键词： Pulmonary disease chronic obstructive HSP27 heat-shock proteins cyclophilin a proteomics