丁基苯酞对沙土鼠全脑缺血再灌注损伤后p-ERK、Caspase-3表达的影响
摘要
前言
缺血性脑卒中具有发病率高、死亡率高、致残率高等特点,因此缺血性脑卒中的防治成为社会和医学界关注的重要课题。缺血再灌注损伤的程度是决定缺血性脑卒中预后的重要因素。近年来关于脑缺血再灌注损伤的发病机制、病理变化及其防治的研究越来越多。丁基苯酞(dl-3n-butylphthalide,NBP)是由中国医学科学院药物研究所研制的,2002年已经被国家食品药品监督管理局批准为治疗缺血性脑卒中的一类新药。已有一些研究表明NBP能够从多个环节改善脑缺血的损伤。
许多研究证明,细胞外信号调节激酶(extracellular signal regulated proteinkinase, ERK)对脑缺血再灌注损伤后细胞的增殖、分化、凋亡起到重要调控作用。而Caspase-3则直接参与脑缺血再灌注损伤后细胞凋亡的发生,是凋亡通路的重要枢纽。
本次实验,我们采用沙土鼠全脑缺血再灌注模型动态观察NBP对脑缺血再灌注损伤后p-ERK、Caspase-3表达的影响,
实验材料和方法
1、动物分组
54只健康雄性沙土鼠(体重60-70g)随机分为3组：假手术组(CON)、缺血再灌注组(I/R)、NBP治疗组(I/R+NBP),每组又分为3个亚组(1d、3d、7d)。假手术组仅游离双侧颈总动脉但不阻断,腹腔注射植物油。缺血再灌注组行双侧颈总动脉钳夹术,缺血后10分钟松开夹子立即腹腔注射植物油。NBP治疗组造模过程同缺血再灌注组,于缺血后10分钟松开夹子立即腹腔注射NBP 40mg/kg。以上各组分别于再灌注1d、3d、7d用Western Blot法检测海马CA1区p-ERK和caspase-3的表达,用Nissl染色法观察海马CA1区神经锥体细胞的变化。
2、沙土鼠全脑缺血再灌注模型制备
采用双侧颈总动脉钳夹术制作沙土鼠全脑缺血再灌注模型
3、Nissl染色
每只沙土鼠从右侧大脑半球分离出海马组织做石蜡切片,石蜡切片再脱蜡后脱经ddH20冲洗,然后放在Nissl染液中染色,结束后两次顺序脱水,最后中性树胶封片。镜下观察海马CA1区神经锥体细胞的变化。
4、Western blot法测沙土鼠海马CA1区p-ERK、Caspase-3
每只沙土鼠从左侧大脑半球分离出海马组织,经称重、制备蛋白样品、行SDS-聚丙烯酰胺凝胶电泳、再转印到PVDF膜上。封闭后分别同多克隆抗磷酸化ERK抗体和兔抗Caspase-3多克隆抗体4℃水平摇床过夜。然后同相应二抗室温下水平摇床1h, TBST洗膜10min×3次,发光显色,用ImageJ对目的条带进行分析。
结果
1、光镜下,与缺血再灌注组相对照,NBP治疗组细胞排列较整齐,细胞大而圆,尼氏体深染,但仍可见少量胞浆浓染。从锥体细胞数的变化上,再灌注组神经元大量减少,不着色,NBP治疗组神经元数量较多(p<0.01)。再灌注3d椎体细胞减少最多,NBP组与再灌注组趋势一致。
2、1d时NBP组较缺血再灌注组p-ERK升高明显,具有统计学意义(p(0.05).3d时两组的表达均有所下降,但NBP组仍明显高于再灌注组(p<0.01).7d时再灌注组的表达再次升高,而NBP组降至同假手术组。两组差别有统计学意义(p<0.01)。
3、1d时NBP治疗组可以降低缺血再灌注后海马区Caspase-3的蛋白表达(p<0.05)。3d时Caspase-3蛋白的升高达高峰,NBP治疗组较再灌注组的表达明显降低(p<0.05)。7d时各组均回落,无统计学意义。
讨论
丁基苯酞(dl-3n-butylphthalide,NBP)是一种专门用于治疗脑血栓的国家级一类药品,多项研究证明该药对缺血后的脑组织具有保护作用。本实验通过Nissl染色从形态学上可以观察到NBP治疗组沙土鼠海马CA1细胞较缺血再灌注组形态完整,排列较整齐,细胞大而圆,尼氏体深染,锥体细胞的丢失减少。结果表明NBP具有减轻沙土鼠全脑缺血再灌注损伤的作用,与既往实验相符。目前一些研究认为ERK的激活与细胞增殖有关,可促进细胞存活。本实验研究证明,NBP组较缺血再灌注组的p-ERK升高明显(p<0.05),提示NBP可能通过调节p-ERK的表达起到保护脑组织的作用。Caspase-3是凋亡过程中的关键蛋白酶,Caspase-3蛋白含量增加,可引发细胞凋亡。本实验研究证明沙土鼠脑缺血再灌注损伤后应用NBP治疗可明显抑制Caspase-3的升高(P<0.05),说明NBP可通过抑制Caspase-3的活性升高而起到保护脑组织的作用。
结论
1、NBP可以改善沙土鼠全脑缺血再灌注后海马CA1区神经细胞的损伤。
2、NBP可以增加沙土鼠全脑缺血再灌注后海马CA1区p-ERK的表达。
3、NBP可以抑制沙土鼠全脑缺血再灌注后海马CA1区Caspase-3的表达。
Preface
Ischemic stroke have a high incidence, high mortality and high disability, so prevention and treatment of ischemic stroke of social and medical attention to an important issue. The extent of ischemia-reperfusion injury of ischemic stroke is an important factor in Prognosis of ischemic stroke. In recent years, research on the pathogenesis、prevention and treatment of Cerebral ischemia-reperfusion is more and more. NBP by the Chinese Academy of Medical Sciences of drugs developed by the Institute in 2002, has been approved by the State Food and Drug Administration for the treatment of ischemic stroke in a class of drugs. Many existing research shows that NBP can improve cerebral ischemic damage from multiple links.
Many studies have shown that, Extracellular signal-regulated kinase (extracellular signal-regulated kinase, ERK) play an important regulatory role of the cerebral ischemia-reperfusion injury in cell proliferation, differentiation and apoptosis. Caspase-3 directly involved in the cerebral ischemia-reperfusion injury in the occurrence of apoptosis, is an important hub for apoptosis pathway.
In this experiment, we have adopted a gerbil model of cerebral ischemia-reperfusion dynamic observation of NBP on the p-ERK, Caspase-3 expression after cerebral ischemia-reperfusion injury.
Materials andMethod
1.Groups
All 54 healthy male gerbils (body weight 60-70g) were randomly divided into 3 groups:Sham-operated group (CON), ischemia-reperfusion group (I/R), NBP treated group (I/R+NBP),Each group is divided into three sub-groups (1d,3d,7d). Sham-operated group, only free, but not bilateral carotid artery occlusion, Intraperitoneal injection of vegetable oil; Ischemia-reperfusion group were applied to bilateral common carotid artery clamp technique, Release the clip immediately after the intraperitoneal injection of vegetable oil after ischemia 10 minutes; NBP treatment group modeling process with the ischemia-reperfusion, Release the clip on the NBP 40mg/kg intraperitoneally immediately after ischemia 10 minutes. Each group using the Western Blot to detect p-ERK, caspase-3 expression in hippocampusin CA1 after the reperfusion 1d,3d,7d.observed hippocampal CA1 neurona pyramidal cells changes by Nissl staining.
2.Preparation of gerbil cerebral ischemia-reperfusion model
Use of bilateral common carotid artery clamp technique produced gerbil model of global cerebral ischemia-reperfusion.
3.Nissl staining
Paraffin sections after dewaxing through ddH2O washing, and then placed in Nissl dye liquor in the dyeing, after the two sequential dehydration, the last neutral gum Fengpian. Observed hippocampal CA1 neuronal pyramidal cells changes in the microscope.
4.Western blot Measuring gerbil hippocampal CA1 area p-ERK, Caspase-3
Each gerbil was isolated from the left cerebral hemisphere, hippocampus, weighing, preparation of protein samples, implementation of SDS-polyacrylamide gel electrophoresis, and then transfer to a PVDF membrane. Closed separately with polyclonal anti-phospho-ERK antibodies and rabbit anti-Caspase-3 polyclonal antibody level of shaking 4℃overnight. And then at room temperature with the appropriate level of two anti-shaker 1h, TBST wash membrane 10min×3 times, luminous, analysis purpose bands with Image J.
Results
1. In hemicroscope, NBP treated group compared with ischemia-reperfusion group, the cells large, round, deeply stained Nissl bodies, but still shows a small amount of cytoplasm stain. From the changes in pyramidal cells on reperfusion a significant reduction in neurons, non-coloring, larger number of neurons in the NBP treatment group (p<0.01);. Vertebral body cell become least when the reperfusion time 3d.NBP group and reperfusion group Trend unanimously.
1. 1d,the NBP group than in the ischemia-reperfusion group p-ERK increased significantly(p<0.05).3 d,the expression of the two groups have decreased, but the NBP group was still significantly higher than reperfusion group (p<0.01).7 d when the reperfusion group, the expression increased again, while the NBP group decreased with the sham-operated group. Difference between the two groups was statistically significant (p<0.01).
2. 1d, the NBP treatment group can be reduced in hippocampus after ischemia-reperfusion Caspase-3 protein expression (p<0.05).3d, Caspase-3 reached the peak, NBP treated group than reperfusion group expression was significantly lower (p<0.05).7d each group were down, no statistical significance.
Disscussion
Butylphthalide (dl-3n-butylphthalide, NBP) is a specialized state-level for the treatment of cerebral thrombosis a class of drugs, a number of studies have shown the drug to the brain tissue after ischemia has a protective effect. By Nissl staining in this experiment can be observed from the morphology of the NBP treatment group compared with gerbil hippocampal CA1 cells in ischemia-reperfusion group intact, arranged more neatly, the cells large, round, deeply stained Nissl bodies, reducing the loss of pyramidal cells. The results show that NBP to relieve cerebral ischemia-reperfusion injury, consistent with past experiments. At present, some studies suggest that ERK activation and cell proliferation, can promote cell survival. This experiment studies have shown that, NBP group than in the ischemia-reperfusion group of p-ERK increased significantly (p＜0.05), prompted NBP may be by regulating the expression of p-ERK play a role in protection of brain tissue. Caspase-3 is the key protease during apoptosis, the experimental studies have shown that NBP treatment can significantly inhibit Caspase-3 elevated (P<0.05), Shows that NBP can play a role in protecting brain tissue by inhibiting the activity of Caspase-3 increased.
Conclusions
1. NBP can improve neuronal cells damage in gerbils hippocampal CA1 after cerebral ischemia and reperfusion
2. NBP can increase the gerbil hippocampal CA1 area p-ERK expression after cerebral ischemia-reperfusion.
3. NBP can inhibit the gerbil hippocampal CA1 area Caspase-3 expression after cerebral ischemia-reperfusion
缺血性脑卒中具有发病率高、死亡率高、致残率高等特点,因此缺血性脑卒中的防治成为社会和医学界关注的重要课题。缺血再灌注损伤的程度是决定缺血性脑卒中预后的重要因素。近年来关于脑缺血再灌注损伤的发病机制、病理变化及其防治的研究越来越多。丁基苯酞(dl-3n-butylphthalide,NBP)是由中国医学科学院药物研究所研制的,2002年已经被国家食品药品监督管理局批准为治疗缺血性脑卒中的一类新药。已有一些研究表明NBP能够从多个环节改善脑缺血的损伤。
许多研究证明,细胞外信号调节激酶(extracellular signal regulated proteinkinase, ERK)对脑缺血再灌注损伤后细胞的增殖、分化、凋亡起到重要调控作用。而Caspase-3则直接参与脑缺血再灌注损伤后细胞凋亡的发生,是凋亡通路的重要枢纽。
本次实验,我们采用沙土鼠全脑缺血再灌注模型动态观察NBP对脑缺血再灌注损伤后p-ERK、Caspase-3表达的影响,
实验材料和方法
1、动物分组
54只健康雄性沙土鼠(体重60-70g)随机分为3组：假手术组(CON)、缺血再灌注组(I/R)、NBP治疗组(I/R+NBP),每组又分为3个亚组(1d、3d、7d)。假手术组仅游离双侧颈总动脉但不阻断,腹腔注射植物油。缺血再灌注组行双侧颈总动脉钳夹术,缺血后10分钟松开夹子立即腹腔注射植物油。NBP治疗组造模过程同缺血再灌注组,于缺血后10分钟松开夹子立即腹腔注射NBP 40mg/kg。以上各组分别于再灌注1d、3d、7d用Western Blot法检测海马CA1区p-ERK和caspase-3的表达,用Nissl染色法观察海马CA1区神经锥体细胞的变化。
2、沙土鼠全脑缺血再灌注模型制备
采用双侧颈总动脉钳夹术制作沙土鼠全脑缺血再灌注模型
3、Nissl染色
每只沙土鼠从右侧大脑半球分离出海马组织做石蜡切片,石蜡切片再脱蜡后脱经ddH20冲洗,然后放在Nissl染液中染色,结束后两次顺序脱水,最后中性树胶封片。镜下观察海马CA1区神经锥体细胞的变化。
4、Western blot法测沙土鼠海马CA1区p-ERK、Caspase-3
每只沙土鼠从左侧大脑半球分离出海马组织,经称重、制备蛋白样品、行SDS-聚丙烯酰胺凝胶电泳、再转印到PVDF膜上。封闭后分别同多克隆抗磷酸化ERK抗体和兔抗Caspase-3多克隆抗体4℃水平摇床过夜。然后同相应二抗室温下水平摇床1h, TBST洗膜10min×3次,发光显色,用ImageJ对目的条带进行分析。
结果
1、光镜下,与缺血再灌注组相对照,NBP治疗组细胞排列较整齐,细胞大而圆,尼氏体深染,但仍可见少量胞浆浓染。从锥体细胞数的变化上,再灌注组神经元大量减少,不着色,NBP治疗组神经元数量较多(p<0.01)。再灌注3d椎体细胞减少最多,NBP组与再灌注组趋势一致。
2、1d时NBP组较缺血再灌注组p-ERK升高明显,具有统计学意义(p(0.05).3d时两组的表达均有所下降,但NBP组仍明显高于再灌注组(p<0.01).7d时再灌注组的表达再次升高,而NBP组降至同假手术组。两组差别有统计学意义(p<0.01)。
3、1d时NBP治疗组可以降低缺血再灌注后海马区Caspase-3的蛋白表达(p<0.05)。3d时Caspase-3蛋白的升高达高峰,NBP治疗组较再灌注组的表达明显降低(p<0.05)。7d时各组均回落,无统计学意义。
讨论
丁基苯酞(dl-3n-butylphthalide,NBP)是一种专门用于治疗脑血栓的国家级一类药品,多项研究证明该药对缺血后的脑组织具有保护作用。本实验通过Nissl染色从形态学上可以观察到NBP治疗组沙土鼠海马CA1细胞较缺血再灌注组形态完整,排列较整齐,细胞大而圆,尼氏体深染,锥体细胞的丢失减少。结果表明NBP具有减轻沙土鼠全脑缺血再灌注损伤的作用,与既往实验相符。目前一些研究认为ERK的激活与细胞增殖有关,可促进细胞存活。本实验研究证明,NBP组较缺血再灌注组的p-ERK升高明显(p<0.05),提示NBP可能通过调节p-ERK的表达起到保护脑组织的作用。Caspase-3是凋亡过程中的关键蛋白酶,Caspase-3蛋白含量增加,可引发细胞凋亡。本实验研究证明沙土鼠脑缺血再灌注损伤后应用NBP治疗可明显抑制Caspase-3的升高(P<0.05),说明NBP可通过抑制Caspase-3的活性升高而起到保护脑组织的作用。
结论
1、NBP可以改善沙土鼠全脑缺血再灌注后海马CA1区神经细胞的损伤。
2、NBP可以增加沙土鼠全脑缺血再灌注后海马CA1区p-ERK的表达。
3、NBP可以抑制沙土鼠全脑缺血再灌注后海马CA1区Caspase-3的表达。
Preface
Ischemic stroke have a high incidence, high mortality and high disability, so prevention and treatment of ischemic stroke of social and medical attention to an important issue. The extent of ischemia-reperfusion injury of ischemic stroke is an important factor in Prognosis of ischemic stroke. In recent years, research on the pathogenesis、prevention and treatment of Cerebral ischemia-reperfusion is more and more. NBP by the Chinese Academy of Medical Sciences of drugs developed by the Institute in 2002, has been approved by the State Food and Drug Administration for the treatment of ischemic stroke in a class of drugs. Many existing research shows that NBP can improve cerebral ischemic damage from multiple links.
Many studies have shown that, Extracellular signal-regulated kinase (extracellular signal-regulated kinase, ERK) play an important regulatory role of the cerebral ischemia-reperfusion injury in cell proliferation, differentiation and apoptosis. Caspase-3 directly involved in the cerebral ischemia-reperfusion injury in the occurrence of apoptosis, is an important hub for apoptosis pathway.
In this experiment, we have adopted a gerbil model of cerebral ischemia-reperfusion dynamic observation of NBP on the p-ERK, Caspase-3 expression after cerebral ischemia-reperfusion injury.
Materials andMethod
1.Groups
All 54 healthy male gerbils (body weight 60-70g) were randomly divided into 3 groups:Sham-operated group (CON), ischemia-reperfusion group (I/R), NBP treated group (I/R+NBP),Each group is divided into three sub-groups (1d,3d,7d). Sham-operated group, only free, but not bilateral carotid artery occlusion, Intraperitoneal injection of vegetable oil; Ischemia-reperfusion group were applied to bilateral common carotid artery clamp technique, Release the clip immediately after the intraperitoneal injection of vegetable oil after ischemia 10 minutes; NBP treatment group modeling process with the ischemia-reperfusion, Release the clip on the NBP 40mg/kg intraperitoneally immediately after ischemia 10 minutes. Each group using the Western Blot to detect p-ERK, caspase-3 expression in hippocampusin CA1 after the reperfusion 1d,3d,7d.observed hippocampal CA1 neurona pyramidal cells changes by Nissl staining.
2.Preparation of gerbil cerebral ischemia-reperfusion model
Use of bilateral common carotid artery clamp technique produced gerbil model of global cerebral ischemia-reperfusion.
3.Nissl staining
Paraffin sections after dewaxing through ddH2O washing, and then placed in Nissl dye liquor in the dyeing, after the two sequential dehydration, the last neutral gum Fengpian. Observed hippocampal CA1 neuronal pyramidal cells changes in the microscope.
4.Western blot Measuring gerbil hippocampal CA1 area p-ERK, Caspase-3
Each gerbil was isolated from the left cerebral hemisphere, hippocampus, weighing, preparation of protein samples, implementation of SDS-polyacrylamide gel electrophoresis, and then transfer to a PVDF membrane. Closed separately with polyclonal anti-phospho-ERK antibodies and rabbit anti-Caspase-3 polyclonal antibody level of shaking 4℃overnight. And then at room temperature with the appropriate level of two anti-shaker 1h, TBST wash membrane 10min×3 times, luminous, analysis purpose bands with Image J.
Results
1. In hemicroscope, NBP treated group compared with ischemia-reperfusion group, the cells large, round, deeply stained Nissl bodies, but still shows a small amount of cytoplasm stain. From the changes in pyramidal cells on reperfusion a significant reduction in neurons, non-coloring, larger number of neurons in the NBP treatment group (p<0.01);. Vertebral body cell become least when the reperfusion time 3d.NBP group and reperfusion group Trend unanimously.
1. 1d,the NBP group than in the ischemia-reperfusion group p-ERK increased significantly(p<0.05).3 d,the expression of the two groups have decreased, but the NBP group was still significantly higher than reperfusion group (p<0.01).7 d when the reperfusion group, the expression increased again, while the NBP group decreased with the sham-operated group. Difference between the two groups was statistically significant (p<0.01).
2. 1d, the NBP treatment group can be reduced in hippocampus after ischemia-reperfusion Caspase-3 protein expression (p<0.05).3d, Caspase-3 reached the peak, NBP treated group than reperfusion group expression was significantly lower (p<0.05).7d each group were down, no statistical significance.
Disscussion
Butylphthalide (dl-3n-butylphthalide, NBP) is a specialized state-level for the treatment of cerebral thrombosis a class of drugs, a number of studies have shown the drug to the brain tissue after ischemia has a protective effect. By Nissl staining in this experiment can be observed from the morphology of the NBP treatment group compared with gerbil hippocampal CA1 cells in ischemia-reperfusion group intact, arranged more neatly, the cells large, round, deeply stained Nissl bodies, reducing the loss of pyramidal cells. The results show that NBP to relieve cerebral ischemia-reperfusion injury, consistent with past experiments. At present, some studies suggest that ERK activation and cell proliferation, can promote cell survival. This experiment studies have shown that, NBP group than in the ischemia-reperfusion group of p-ERK increased significantly (p＜0.05), prompted NBP may be by regulating the expression of p-ERK play a role in protection of brain tissue. Caspase-3 is the key protease during apoptosis, the experimental studies have shown that NBP treatment can significantly inhibit Caspase-3 elevated (P<0.05), Shows that NBP can play a role in protecting brain tissue by inhibiting the activity of Caspase-3 increased.
Conclusions
1. NBP can improve neuronal cells damage in gerbils hippocampal CA1 after cerebral ischemia and reperfusion
2. NBP can increase the gerbil hippocampal CA1 area p-ERK expression after cerebral ischemia-reperfusion.
3. NBP can inhibit the gerbil hippocampal CA1 area Caspase-3 expression after cerebral ischemia-reperfusion
引文
1 Ma XL, Kumar S, Gao F, et al.Inhibition of p38 mitogen activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion [J].Circulation,1999,99(13):1685-1691.
2 Salvesen GS.Caspases and apoptosis[J].Essays Biochem,2002,38:9-19.
3 Wu DC,Ye W,Che XM,et al.Actiration of mitogen-actibated protein kinases after permanent cerebral artery lcclusion in mouse brain[J].Cereb Blood Flow Merab,2000,20:1320-1330.
4 Stennieke HR, Salvesen GS.ProPerties of caspases[J].Bioehim BioPhys Aets,1998, 1387(1-2):17-31.
5 ChenJ, NagayamaT, JinK, et al.Indution of Caspase-3 like Protease may mediate delayed neuronal death in the hiPPoeamPus after trainsient cerebralisehemia[J]. Neurosie,1998, 18(13):4914.
6 徐皓亮，冯亦璞。丁基苯酞对大鼠血栓形成及血小板功能的影响。药学学报,2001,36.(5)：329-333。
7 董高翔,冯亦璞。丁基苯酞对局部脑缺血再灌注大鼠脑线粒体ATP-ase抗氧化酶活性和脂质过氧化的影响。中国医学科学院学报,2002,24(1)：93-97。
8 Horstmann S.et al.Actibation of p38 mitogen-activated protein kinase promote neuronal survival in vitro[J]. Neurosci Res.1998 May 15;52 (4);483-490.
9 Wieloch T,Hu BR,Boris-Moiler A,et al.Intracellular signal transduction in the postischemic brain[J].Adv Nerrol 1996;71;371-387.
10 Nozaki K,Nishimura M,Hashimoto N.Mitogen activated protein kinases and cerebral ischemia[J].Mol.Neurobiol2001p;23(1);1-19.
11 Gu Zl,Jiang Z,ZHANG GY,et al.D/phosphorylation of extracellular signal regulated kinases and c-Jun N-rerminal protein kinase in brain ischemic tolerance in rat.Brain Res 2000;860(t-2);157-160.
12 Irving EA,Barone FC,Reith AD,et al. Differential activation of MAPK/ERK and P38/SAPK in neurons and glia following focal cerebra ischemia in the rat [J].Brain Res Mol Brain Res 2000;77(1);65-75.
13 Hicks SD.Parmele KT.Defranco DB.et al.Hypoithermia differentially increase extracellular signal regulated kinase and srress activared protein kinase/C-jun terminal kinase activation in the hi ppocampus during reperfusion after asphyriao cardiae arrest [J].Neuroscience 2000;92 (4); 677-685.
14 Li F,Omori N,Sato K,et al.Coordinate expression of survival p-ERK and proapoprotic eytochrome signals in rat brain neurons after transient MCAO[J].Brain Research,2002,958:83-88.
15 吕建瑞,马宏钟,薛荣亮。前脑缺血再灌注损伤后ERK在大鼠海马的表达及热休克预处理对其表达的影响。第四军医大学学报2006；27(16).
16 Daniel PT.Disseeting the Pathways to death [J].Leukemia,2000,14:2035-2044.
17 Janieke RU, Ng P, SPrenggart ML, et al.CasPase-3 is required for fodrin cleavage but disPensable for cleavage of other death substrate in aPoP tosis.J Biol Chem.1998:273 (25):15510-15545.
18郭颂,饶明俐。Caspase及细胞色素-C在海人藻酸所致海马组织兴奋性毒性损伤中的作用。J Apoplexy and Nervous Diseases,2000,17(5):258-260.
19 Chen J, Nagayama T, Jin K, et al.Indution of CasPase-3 like Protease mayediate delayed neuronal death in the hiPPoeampus after trainsient cerebral isehemia[J]. Neurosie,1998, 18(13):4 914.
20 Chon g ZZ,Feng YP. Effects of dl-3-n-butylphthalide on production of TXB2 and reperfusion[J].Acta Pharmacol Sin,1997,18(6):505-508.
21 LI Q-fu,KONG Shuang-yan,DEJI Qu-Zong,HE Li,ZHOU Dong.Effects of dl-3-n-butylphthalide on Expression of VEGF and bFGF in Rat Brain with Permanent Focal Cerebral. West China Hospitl,2008;39(1):84-88.
22熊杰冯亦璞。丁基苯酞对局灶型脑缺血再灌大鼠脑hsp70mRNA和c-Fos时相表达的影响。药学学报,1998,33(6)：401-406
23熊杰,冯亦璞。丁基苯酞对局灶性脑缺血过程中线粒体损伤的保护作用[J].药学学报,2000,35：408-412.
24 Alessandrini A,Namura S,Mokowitz MA,et al.MEKl protein kinase inhibition proteces against damage resulting from focal cerebral,ischemia.Proc Nail Acad Sci USA 1999:12866-12863.
1 Sweeney MI, Yager JY, Wolzw, et al.Cellularmechanisms involved in brain ischemia[J].Can J Physiol Pharmacol,1995,73(11):1525-1535.
2 Davis RJ.The mitogen-activated protein kinase signal transduction pathway[J].J Biol Chem, 1993,268:14553.
3 彭黎明,王曾礼。细胞凋亡的信号传导机制·细胞凋亡的基础与临床[M]。北京：人民卫生出版社,2000.40-46.
4 徐勇.MAPK家族与糖尿病并发症[J].国外医学内分泌学分册,2001,(1)：8-10.
5 牟金叶,陈晓光.促分裂原激活的蛋白激酶(MAPK)信号传导通路的研究进展[J]·生命科学,2002,208-221.
6 Foorce T, Bonventre J.Growth factors and mitogen-activated protein kinase [J].Hypertension, 1998,31:152-161.
7 Glogowshi EA, Tsiani E, Zhou X, et al.High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1 [J].Kidney Int,1999,55:486-499.
8 陈良恩.JNK信号转导通路及其在应激中的作用[J].国外医学生理病理学与临床分册,2001,21(3)：169-171.
9 Brewster JL, De Valoir T, Dwyer NG, et al.An osmosensing signal transduction pathway in yeast [J].Science,1993,259:1760-1763.
10 Ailawa R, Komuro I, Yamazaki T, et al.Oxidation stress activates extracellular signal-regulated kinases throngh Src and Ras in cultured cardiac monocytes of neonatal rats [J].J Clin Invest,1997,100:1813-1821.
11 Ma XL, Kumar S, Gao F, et al.Inhibition of p38 mitogen activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion [J].Circulation,1999,99(13):1685-1691.
12 Elaine A, Irving EA, Mark Bamford.Role of mitogen and stress-activated kinases in ischemic injury [J].J Cereb Blood FlowMetab,2002,22:631-647.
13 Vanhoutte P, Barnier JV, Guibert B, et al.Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices[J].Mol Cell Bi-ol,1997,19(l):136-146.
14 SchwarzschildMA, Cole RL, MeyersMA, et al.Contrasting calcium dependencies of SAPK and ERK activations by glutamate in cultured striatal neurons[J].J Neurochem,1999,72(6): 2248-2255.
15 Lipto SA, Roserberg PA.Excitatory amino acids as a final common pathway for neurologic disorders [J].N Engl T Med,1994,330:613-622.
16 Singer CA, FigueroaMasot XA, Batchelor RH, et al.The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons [J].J Neurosci,1999,19(7):2455-2463.
17 Walton KM, DiRocco R, Bartlett BA, et al.Activation of p38MAPK in microglia after ischemia [J]. J Neurochem,1998,70(4):1764-1767.
18 Herdegen T, Claret FX, Kallunki T, et al.Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury[J].neurosci,1998,18(14): 5124-5135.
19 Ozawa H, Shioda S, Dohi K, et al.Delayed neuronal cell death in the rat hippocampus is mediated by the mitogen-activated protein kinase signal transduction pathway [J].Neurosci Letters,1999,262:57-60.
20 Oh SW, AhnYM, KangUG, et al.Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock[J].Neurosci Letters,1999, 271:101-104.
21 Namura S, Iihara K, Takama S, et al. Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia[J].Proc Natl Acad Sci USA,2001,98:11569-1
22 Hu BR, Liu CL, Park DJ.Alteration ofMAP kinase pathways after transient forebrain ischemia [J].J Cereb Blood FlowMetab,2000,20:1089-1095.
23 Stanciu M, WangY, KentorR, et al.Persistent activation of ERK contributes to glutamate-induced oxidative in aneuronal cell line and primary cortical neuronal cultures [J]. J Biol Chem,2000,275:12200-12206.
24 Irving EA, Ray AM, Staton PC, et al.Neuroprotection with the MEK inhibitor U0126 following ischemic injury [J]. J Cereb Blood Flow Metab,2001,21(Suppl 1):S379.
25 Wu DC et al. Aetivation of mitogen-aetivated portein kinases atfer per-manent cerebral artery occlusion in mouse brain.J Cereb Blood Flow Metab.2000 Sep;20(9):1320-30.
26 Rving EA, Barone FC, Reith AD, et al.Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat[J].Mol Brain Res,2000, 77:65-75.
27 Hicks SD et al.Hypothermia diefferntialy incerases extarcellular signal-reuglated kinase and srtess-aetivated portein kinas/ee-Jun temrinal kinase activation in the hippocampus during reperfusion after asphuxial cardiac arrest.Neuroscience.200;98(4):677-85
28 Wang X, Wang H, Xu L, et al.Significant neuroprotection against ischemic brain injury by inhibition of the MEK1 protein kinase in mice:exploration of potential mechanism associatedwith apoptosis [J].J Pharmacol Exp Ther,2003,304(1):172-178
29 Alessandrini A, Namura S, Moskowitz MA, et al.MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia[J].Proc Natl Acad Sci USA,1999, 96(22):12866-12869.
30 Pereira DB, Carvalho AP, Duarte CB.Non-specific effects of the MEK inhibitors PD98059 and U0126 on glutamate release from hippocampal synaptosomes[J].Neuropharmacology, 2002,42:9-19.
2 Salvesen GS.Caspases and apoptosis[J].Essays Biochem,2002,38:9-19.
3 Wu DC,Ye W,Che XM,et al.Actiration of mitogen-actibated protein kinases after permanent cerebral artery lcclusion in mouse brain[J].Cereb Blood Flow Merab,2000,20:1320-1330.
4 Stennieke HR, Salvesen GS.ProPerties of caspases[J].Bioehim BioPhys Aets,1998, 1387(1-2):17-31.
5 ChenJ, NagayamaT, JinK, et al.Indution of Caspase-3 like Protease may mediate delayed neuronal death in the hiPPoeamPus after trainsient cerebralisehemia[J]. Neurosie,1998, 18(13):4914.
6 徐皓亮，冯亦璞。丁基苯酞对大鼠血栓形成及血小板功能的影响。药学学报,2001,36.(5)：329-333。
7 董高翔,冯亦璞。丁基苯酞对局部脑缺血再灌注大鼠脑线粒体ATP-ase抗氧化酶活性和脂质过氧化的影响。中国医学科学院学报,2002,24(1)：93-97。
8 Horstmann S.et al.Actibation of p38 mitogen-activated protein kinase promote neuronal survival in vitro[J]. Neurosci Res.1998 May 15;52 (4);483-490.
9 Wieloch T,Hu BR,Boris-Moiler A,et al.Intracellular signal transduction in the postischemic brain[J].Adv Nerrol 1996;71;371-387.
10 Nozaki K,Nishimura M,Hashimoto N.Mitogen activated protein kinases and cerebral ischemia[J].Mol.Neurobiol2001p;23(1);1-19.
11 Gu Zl,Jiang Z,ZHANG GY,et al.D/phosphorylation of extracellular signal regulated kinases and c-Jun N-rerminal protein kinase in brain ischemic tolerance in rat.Brain Res 2000;860(t-2);157-160.
12 Irving EA,Barone FC,Reith AD,et al. Differential activation of MAPK/ERK and P38/SAPK in neurons and glia following focal cerebra ischemia in the rat [J].Brain Res Mol Brain Res 2000;77(1);65-75.
13 Hicks SD.Parmele KT.Defranco DB.et al.Hypoithermia differentially increase extracellular signal regulated kinase and srress activared protein kinase/C-jun terminal kinase activation in the hi ppocampus during reperfusion after asphyriao cardiae arrest [J].Neuroscience 2000;92 (4); 677-685.
14 Li F,Omori N,Sato K,et al.Coordinate expression of survival p-ERK and proapoprotic eytochrome signals in rat brain neurons after transient MCAO[J].Brain Research,2002,958:83-88.
15 吕建瑞,马宏钟,薛荣亮。前脑缺血再灌注损伤后ERK在大鼠海马的表达及热休克预处理对其表达的影响。第四军医大学学报2006；27(16).
16 Daniel PT.Disseeting the Pathways to death [J].Leukemia,2000,14:2035-2044.
17 Janieke RU, Ng P, SPrenggart ML, et al.CasPase-3 is required for fodrin cleavage but disPensable for cleavage of other death substrate in aPoP tosis.J Biol Chem.1998:273 (25):15510-15545.
18郭颂,饶明俐。Caspase及细胞色素-C在海人藻酸所致海马组织兴奋性毒性损伤中的作用。J Apoplexy and Nervous Diseases,2000,17(5):258-260.
19 Chen J, Nagayama T, Jin K, et al.Indution of CasPase-3 like Protease mayediate delayed neuronal death in the hiPPoeampus after trainsient cerebral isehemia[J]. Neurosie,1998, 18(13):4 914.
20 Chon g ZZ,Feng YP. Effects of dl-3-n-butylphthalide on production of TXB2 and reperfusion[J].Acta Pharmacol Sin,1997,18(6):505-508.
21 LI Q-fu,KONG Shuang-yan,DEJI Qu-Zong,HE Li,ZHOU Dong.Effects of dl-3-n-butylphthalide on Expression of VEGF and bFGF in Rat Brain with Permanent Focal Cerebral. West China Hospitl,2008;39(1):84-88.
22熊杰冯亦璞。丁基苯酞对局灶型脑缺血再灌大鼠脑hsp70mRNA和c-Fos时相表达的影响。药学学报,1998,33(6)：401-406
23熊杰,冯亦璞。丁基苯酞对局灶性脑缺血过程中线粒体损伤的保护作用[J].药学学报,2000,35：408-412.
24 Alessandrini A,Namura S,Mokowitz MA,et al.MEKl protein kinase inhibition proteces against damage resulting from focal cerebral,ischemia.Proc Nail Acad Sci USA 1999:12866-12863.
1 Sweeney MI, Yager JY, Wolzw, et al.Cellularmechanisms involved in brain ischemia[J].Can J Physiol Pharmacol,1995,73(11):1525-1535.
2 Davis RJ.The mitogen-activated protein kinase signal transduction pathway[J].J Biol Chem, 1993,268:14553.
3 彭黎明,王曾礼。细胞凋亡的信号传导机制·细胞凋亡的基础与临床[M]。北京：人民卫生出版社,2000.40-46.
4 徐勇.MAPK家族与糖尿病并发症[J].国外医学内分泌学分册,2001,(1)：8-10.
5 牟金叶,陈晓光.促分裂原激活的蛋白激酶(MAPK)信号传导通路的研究进展[J]·生命科学,2002,208-221.
6 Foorce T, Bonventre J.Growth factors and mitogen-activated protein kinase [J].Hypertension, 1998,31:152-161.
7 Glogowshi EA, Tsiani E, Zhou X, et al.High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1 [J].Kidney Int,1999,55:486-499.
8 陈良恩.JNK信号转导通路及其在应激中的作用[J].国外医学生理病理学与临床分册,2001,21(3)：169-171.
9 Brewster JL, De Valoir T, Dwyer NG, et al.An osmosensing signal transduction pathway in yeast [J].Science,1993,259:1760-1763.
10 Ailawa R, Komuro I, Yamazaki T, et al.Oxidation stress activates extracellular signal-regulated kinases throngh Src and Ras in cultured cardiac monocytes of neonatal rats [J].J Clin Invest,1997,100:1813-1821.
11 Ma XL, Kumar S, Gao F, et al.Inhibition of p38 mitogen activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion [J].Circulation,1999,99(13):1685-1691.
12 Elaine A, Irving EA, Mark Bamford.Role of mitogen and stress-activated kinases in ischemic injury [J].J Cereb Blood FlowMetab,2002,22:631-647.
13 Vanhoutte P, Barnier JV, Guibert B, et al.Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices[J].Mol Cell Bi-ol,1997,19(l):136-146.
14 SchwarzschildMA, Cole RL, MeyersMA, et al.Contrasting calcium dependencies of SAPK and ERK activations by glutamate in cultured striatal neurons[J].J Neurochem,1999,72(6): 2248-2255.
15 Lipto SA, Roserberg PA.Excitatory amino acids as a final common pathway for neurologic disorders [J].N Engl T Med,1994,330:613-622.
16 Singer CA, FigueroaMasot XA, Batchelor RH, et al.The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons [J].J Neurosci,1999,19(7):2455-2463.
17 Walton KM, DiRocco R, Bartlett BA, et al.Activation of p38MAPK in microglia after ischemia [J]. J Neurochem,1998,70(4):1764-1767.
18 Herdegen T, Claret FX, Kallunki T, et al.Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury[J].neurosci,1998,18(14): 5124-5135.
19 Ozawa H, Shioda S, Dohi K, et al.Delayed neuronal cell death in the rat hippocampus is mediated by the mitogen-activated protein kinase signal transduction pathway [J].Neurosci Letters,1999,262:57-60.
20 Oh SW, AhnYM, KangUG, et al.Differential activation of c-Jun N-terminal protein kinase and p38 in rat hippocampus and cerebellum after electroconvulsive shock[J].Neurosci Letters,1999, 271:101-104.
21 Namura S, Iihara K, Takama S, et al. Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia[J].Proc Natl Acad Sci USA,2001,98:11569-1
22 Hu BR, Liu CL, Park DJ.Alteration ofMAP kinase pathways after transient forebrain ischemia [J].J Cereb Blood FlowMetab,2000,20:1089-1095.
23 Stanciu M, WangY, KentorR, et al.Persistent activation of ERK contributes to glutamate-induced oxidative in aneuronal cell line and primary cortical neuronal cultures [J]. J Biol Chem,2000,275:12200-12206.
24 Irving EA, Ray AM, Staton PC, et al.Neuroprotection with the MEK inhibitor U0126 following ischemic injury [J]. J Cereb Blood Flow Metab,2001,21(Suppl 1):S379.
25 Wu DC et al. Aetivation of mitogen-aetivated portein kinases atfer per-manent cerebral artery occlusion in mouse brain.J Cereb Blood Flow Metab.2000 Sep;20(9):1320-30.
26 Rving EA, Barone FC, Reith AD, et al.Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat[J].Mol Brain Res,2000, 77:65-75.
27 Hicks SD et al.Hypothermia diefferntialy incerases extarcellular signal-reuglated kinase and srtess-aetivated portein kinas/ee-Jun temrinal kinase activation in the hippocampus during reperfusion after asphuxial cardiac arrest.Neuroscience.200;98(4):677-85
28 Wang X, Wang H, Xu L, et al.Significant neuroprotection against ischemic brain injury by inhibition of the MEK1 protein kinase in mice:exploration of potential mechanism associatedwith apoptosis [J].J Pharmacol Exp Ther,2003,304(1):172-178
29 Alessandrini A, Namura S, Moskowitz MA, et al.MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia[J].Proc Natl Acad Sci USA,1999, 96(22):12866-12869.
30 Pereira DB, Carvalho AP, Duarte CB.Non-specific effects of the MEK inhibitors PD98059 and U0126 on glutamate release from hippocampal synaptosomes[J].Neuropharmacology, 2002,42:9-19.