脑缺血再灌注降低大脑皮质中前蛋白转化酶2的活性

doi:10.3969/j.issn.1673-5374.2013.01.011

中国神经再生研究(英文版) ›› 2013, Vol. 8 ›› Issue (1): 83-89.doi: 10.3969/j.issn.1673-5374.2013.01.011

• 原著:脑损伤修复保护与再生 • 上一篇下一篇

脑缺血再灌注降低大脑皮质中前蛋白转化酶2的活性

收稿日期:2012-08-17 修回日期:2012-12-03 出版日期:2013-01-05 发布日期:2013-01-05

Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation

Shuqin Zhan^{1, 2}, An Zhou², Chelsea Piper², Tao Yang²

1 Department of Neurology, the Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China
2 Robert S. Dow Neurobiology Laboratories, Legacy Clinic Research and Technology Center, Portland, OR 97232, USA

Received:2012-08-17 Revised:2012-12-03 Online:2013-01-05 Published:2013-01-05
Contact: Shuqin Zhan, Department of Neurology, the Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China; Robert S. Dow Neurobiology Laboratories, Legacy Clinic Research and Technology Center, Portland, OR 97232, USA, zhanshuqin@163.com.
About author:Shuqin Zhan☆, M.D., Ph.D., Associate chief physician.
Supported by:
The project was financially supported by the National Natural Science Foundation of China, No. 81070999; the foundation of Xi’an Jiaotong University, No. 95, 2009; Foundation of the Second Affiliated Hospital of Xi’an Jiaotong University, No. RC (GG) 201109; the US National Institutes of Health, No. NS046560; and the American Heart Association, No. 0450142Z.

摘要/Abstract

摘要：

以大脑中动脉阻塞及缺氧缺糖方法建立脑缺血再灌注模型和缺氧缺糖脑皮质神经元模型。结果表明脑缺血再灌注大鼠皮质前蛋白转化酶2活性减少，且随再灌注时间的延长大鼠皮质前蛋白转化酶2活性降低；而体外缺血缺氧培养的大鼠皮质神经元中TUNEL阳性神经元的数量明显增加，且皮质神经元中前蛋白转化酶2的活性也明显下降，也随缺血缺氧培养时间的延长，皮质神经元中前蛋白转化酶2的活性继续下降。提示缺血再灌注及缺氧缺糖引起的大鼠脑皮质及培养的大鼠皮质神经元的前蛋白转化酶2活性的衰减可能在脑损伤中具有重要的病理作用。

关键词: 神经再生, 脑损伤, 前蛋白转化酶2, 皮质, 神经元, 脑缺血再灌注, 缺氧缺糖, 体内研究, 体外研究, 基金资助文章, 图片文章

Abstract:

In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.

Key words: neural regeneration, brain injury, proprotein convertase 2, cortex, neuron, cerebral ischemia/reperfusion, oxygen-glucose deprivation, in vivo study, in vitro study, grants-supported paper, photographs-containing paper, neuroregeneration

. 脑缺血再灌注降低大脑皮质中前蛋白转化酶2的活性[J]. 中国神经再生研究(英文版), 2013, 8(1): 83-89.

Shuqin Zhan, An Zhou, Chelsea Piper, Tao Yang. Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation[J]. Neural Regeneration Research, 2013, 8(1): 83-89.

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引言

材料方法

Design

A randomized controlled animal study focusing on cytobiology.

Time and setting

The experiment was performed at the Robert S. Dow Neurobiology Laboratories, Legacy Clinical Research and Technology Center, Portland, OR, USA from March 2006 to July 2007.

Materials

A total of 18 male Sprague-Dawley rats, weighing 250–300 g, of clean grade, aged 3 months, were purchased from Charles River Laboratories (Wilmington, MA, USA). Timed pregnant rats were also purchased from Charles River Laboratories. About 30 rat pups born within 3 days were used for cell cultures. The rats were housed on a 12-hour light/dark cycle and freely fed at 22 ± 2°C and a relative humidity of 55 ± 5%. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Methods

Modeling of transient focal cerebral ischemia/ reperfusion

Focal cerebral ischemia/reperfusion was induced by middle cerebral artery occlusion^[32-34]. Briefly, rats were anesthetized with 4% isoflurane in 70% nitrous oxide/ 30% oxygen, and maintained with 2% isoflurane in 70% nitrous oxide/30% oxygen. Middle cerebral artery occlusion was achieved by introducing a 3-0 silk suture into the lumen of the right internal carotid artery, with the external carotid artery and the extracranial branch of the internal carotid artery ligated. After 100 minutes of middle cerebral artery occlusion, the suture was withdrawn to allow reperfusion up to 24 hours. Control animals were sham operated. They underwent the same surgical procedure, but the suture was not advanced to the middle cerebral artery. Relative regional cerebral blood flow was monitored with a laser-Doppler flowmetry apparatus (Transonic Systems Inc., Ithaca, NY, USA), and successful occlusion and reperfusion were confirmed using this system. A ≥ 80% reduction in regional cerebral blood flow observed immediately after introducing the silk suture into the lumen of the internal carotid artery was required in the current study^[35].

Preparation of cortical tissues

At 8 and 24 hours after reperfusion, animals were decapitated under anesthesia. Tissues from the ischemic cortical regions of the territory supplied by the middle cerebral artery, as illustrated in earlier studies^{[32-33, 36]}, were immediately dissected, frozen on dry ice, and stored at –80°C.

Primary rat cortical neuron cultures and in vitro simulated ischemia

Primary rat cortical neurons were cultured as follows. Briefly, the cerebral cortices from about 30 rat pups born within 3 days were anesthetized with isoflurane and dissected and incubated with 0.05% ethylenediaminetetraacetic acid in PBS for 10 minutes at 37°C, followed by trituration with flame-polished glass pipettes. For neuronal cultures, dispersed cortical cells were seeded onto poly-L-ornithine-coated 35-mm dishes at a density of 1 × 10⁶ cells/dish. Cells were maintained in Eagle’s minimum essential medium supplemented with 10% horse serum (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO₂. 5-fluoro-2-deoxyuridine (5 µM) and uridine (5 µM) were added 72 hours after seeding to suppress the growth of glial cells. Cells were used for experiments 7 to 8 days after seeding^[37].

Ischemia was simulated in cultured neurons by oxygen- glucose deprivation, as previously described^[32] , by incubating cells in glucose-free, serum-free, and glutamine-free medium in an anaerobic chamber (Forma Scientific, Marietta, OH, USA) containing 85% nitrous oxide/5% carbon dioxide/10% hydrogen for 30, 60, 120 or 180 minutes. Cells in the control group were incubated in control medium (consisting of 25 mM Hepes, pH 7.4; 2 mM CaCl₂; 135 mM NaCl; 5 mM KCl; 1 × essential amino acids; 1 × L-glutamine; 20 mM glucose; penicillin/streptomycin) for 30, 60, 120 or 180 minutes. After oxygen-glucose deprivation and control medium treatment, cells were allowed to recover in complete medium (Neurobasal-A with Glutamax-1, B27 supplement, Pen/Strep) under normal conditions overnight.

TUNEL staining for neuronal apoptosis

The oxygen-glucose deprivation-injured cells were identified by detecting DNA fragmentation using the TUNEL method with a commercial kit (Roche Diagnostics GmbH, Roche Applied Science, 68298 Mannheim, Germany), following the manufacturer’s instructions. Briefly, cell samples (approximately 360 cells in a field at 100 × magnification) were fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4). Samples were rinsed with PBS and incubated in 0.1% Triton X-100 in PBS for 2 minutes on ice (2–8°C). Samples were then rinsed twice with PBS, and a 50-µL aliquot of TUNEL reaction mixture was added to the samples, followed by incubation in a humidified atmosphere for 60 minutes at 37°C in the dark. Samples were rinsed with PBS (three times) and mounted with DAPI-containing mounting solution. After staining, fluorescence signals were examined with a Leica epifluorescence microscope (Leica Microsystems Inc., Bannockburn, IL, USA) attached to a digital camera, and analyzed with the assistance of the Bioquant program (Bioquant Image Analysis, Nashville, TN, USA). TUNEL-positive cells were counted in ten fields at 100 × magnification and summed for each dish.

Proprotein convertase 2 activity assay

Proteins were extracted from brain tissues following standard protocols^[38]. The extraction buffer for cortical samples and cells consisted of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 1% TritonX-100, 0.1% sodium deoxycholate, 0.1% SDS, 2 mM edetic acid, and a cocktail of protease inhibitors^[39]. Insoluble material was removed by centrifugation at 10 000 × g for 10 minutes at 4°C. Analyses of proprotein convertase 2 activity followed protocols described by Berman et al ^[39]. Briefly, 20 mg of proteins from tissue homogenates were incubated with 200 mM L-pGlu-Arg-Thr-Lys-Arg-7- amino-4-methylcoumarin (Peptides International, Louisville, KY, USA) in 100 mM sodium acetate, pH 5.0, and 1 mM CaCl₂ at 37°C for 4 hours. In parallel incubations, 1 mM carboxyl-terminal peptide (Sigma- Genosys, The Woodlands, TX, USA), a proprotein convertase 2-specific inhibitor^[40], was added to reactions. The release of 7-amino-4-methylcoumarin (AMC) was measured using a Spectra Max GEMINI spectrofluorometer (Molecular Devices, Union City, CA, USA; excitation 360 nm/emission 480 nm). The amount of product formed was calculated using free AMC as a standard. The activity inhibited by the presence of carboxyl-terminal peptide was considered as a proprotein convertase 2-specific activity.

Statistical analysis

SPSS 12.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Data were expressed as mean ± SEM. Homogeneity of variance test was performed. Changes of proprotein convertase 2 activity at multiple reperfusion hours were compared using a randomized design one-way analysis of variance. For post-hoc testing, the least significant difference t-test was used for comparison between groups. The percentages of TUNEL-positive cells were compared between the control and oxygen-glucose deprivation groups using the paired t-test, where a value of P < 0.05 was accepted as significant.

讨论

文章快阅

延伸阅读

1中文内容介绍：
神经肽的内蛋白水解是由前蛋白转化酶完成的，前蛋白转化酶2是前蛋白转化酶家族的一员，目前已确认的哺乳动物前蛋白转化酶有9种：前蛋白转化酶1、前蛋白转化酶2、Furin、前蛋白转化酶4、前蛋白转化酶5、PACE4、前蛋白转化酶7、SKI-1 (或S1P或前蛋白转化酶8)及PCSK9/NARC-1(或前蛋白转化酶9)。前蛋白转化酶对应激高度敏感，其激活、成熟及起作用需要严格的细胞状态，脑缺血时钙从细胞外快速内流及从线粒体、内质网快速外流，伴有组织酸中毒，因此我们有理由认为脑缺血引起的细胞状态的改变不能维持稳定的神经肽加工内环境，干扰前蛋白转化酶2的激活及作用。国内外既往有关前蛋白转化酶2的研究多集中在其与内分泌的关系上，很少有人报道前蛋白转化酶2在脑缺血方面的变化及作用，为观察局灶性脑缺血大鼠脑皮质中及培养的缺氧缺糖大鼠脑皮质神经元中前蛋白转化酶2活性的经时变化，因此进行了此项研究。

用大脑中动脉阻塞的方法建立短暂性局灶性脑缺血模型，在100min大脑中动脉阻塞后再灌注达24 h，在每个再灌注结束时间点8及24 h，假手术对照组及大脑中动脉阻塞大鼠在麻醉状态下被断头取脑，取缺血脑皮质行前蛋白转化酶2活性测定；用培养的缺氧缺糖大鼠脑皮质神经元建立离体的短暂性局灶性脑缺血模型，缺氧缺糖 30，60，120及180 min后换常规培养液培养箱中过夜后TUNEL染色行凋亡细胞分析并前蛋白转化酶2活性测定。随缺血及缺氧缺糖时间的延长，大鼠脑皮质及培养的大鼠皮质神经元的前蛋白转化酶2活性逐渐降低，缺血及缺氧缺糖引起的大鼠脑皮质及培养的大鼠皮质神经元的前蛋白转化酶2活性的降低将影响它对神经肽的加工作用，进而影响其底物神经肽对缺血神经元的作用。

2实验设计及文章构思：
近年已观察了前蛋白转化酶家族中的前蛋白转化酶1、前蛋白转化酶2及前蛋白转化酶枯草溶菌素9在脑缺血时的它们的mRNA表达水平及前蛋白转化酶1、前蛋白转化酶2蛋白表达水平的经时表达变化，研究表明100min大脑中动脉阻塞后缺血侧脑皮质前蛋白转化酶1mRNA及前蛋白转化酶1的表达在再灌注4，8及16h逐渐下降，前蛋白转化酶1mRNA在再灌注24h时下降最显著，缺血侧脑皮质前蛋白转化酶1表达水平在再灌注24h较再灌注4，8及16h增高，而此时前蛋白转化酶1mRNA表达水平仍低。大鼠100 min大脑中动脉阻塞后再灌注，前蛋白转化酶2mRNA表达水平在再灌注早期时间段短暂上调，此时的前蛋白转化酶2蛋白表达水平也较高(用免疫细胞化学和蛋白质印迹法测定)，脑组织中前蛋白转化酶2的底物强啡肽A(1-8)的水平在再灌注早期时间段也降低, 给小鼠脑室内注射合成的强啡肽A(1-8)可显著减少缺血性脑损伤的范围，且前蛋白转化酶2敲除小鼠更易产生缺血性脑损伤。大鼠100min大脑中动脉阻塞后再灌注4，8及24h大鼠缺血侧脑皮质内前蛋白转化酶枯草溶菌素9mRNA表达随再灌注时间延长而增高。

3文章学术水平与国内外同类研究的比较：

前蛋白转化酶2是前蛋白转化酶家族中的一员，国内外既往有关前蛋白转化酶2的研究多集中在其与内分泌的关系上，而其中有关前蛋白转化酶2活性研究极少，除Li等[1]发表的有关前蛋白转化酶2在内分泌细胞及内分泌组织活性的测定及Tang等[2]发表的前蛋白转化酶2活性在RGC-5细胞及NS20Y细胞的变化外，国内外极少有其他相关报道。而有关前蛋白转化酶2在脑缺血方面的变化及作用，除作者已发表的前蛋白转化酶2与其底物神经肽在脑缺血时的变化及作用[3]外，国内外未见他人报道前蛋白转化酶2在脑缺血方面的变化及作用, 因此研究领域、理论假设及结果具有创新性。

[1] Li QL, Naqvi S, Shen X, et al. Prohormone convertase 2 enzymatic activity and its regulation in neuro-endocrine cells and tissues. Regul Pept. 2003;110(3):197-205
[2] Tang SS, Zhang JH, Liu HX, et al. Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia. Neurosci Bull. 2009;25(1):7-14.
[3] Zhan S, Zhao H , J White A, et al. Defective neuropeptide processing and ischemic brain injury: a study on proprotein convertase 2 and its substrate neuropeptide in ischemic brains. Journal of Cerebral Blood Flow & Metabolism 2009;29:698-706。

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脑缺血再灌注降低大脑皮质中前蛋白转化酶2的活性

Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation

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