3—硝基丙酸预处理及其神经保护机制

近年来的研究发现，小剂量线粒体毒素3—硝基丙酸预处理可以诱导脑对缺血缺氧产生耐受性。文章就3—硝基丙酸预处理的特点及其神经保护机制的研究进展进行了综述。     

3-硝基丙酸预处理及其神经保护机制

近年来的研究发现,小剂量线粒体毒素3-硝基丙酸预处理可以诱导脑对缺血缺氧产生耐受性。文 章就3-硝基丙酸预处理的特点及其神经保护机制的研究进展进行了综述。     

3-硝基丙酸预处理对大鼠局灶性脑缺血caspase-3活性和神经细胞凋亡的影响

目的 探讨小剂量线粒体毒素3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血脑梗死体积、脑缺血半暗带半胱天冬酶一3(caspase-3)活性和神经细胞凋亡的影响。方法 大鼠腹腔注射3-NPA 3d后制作大脑中动脉闭塞模型，采用TTC染色、TuNEL法、流式细胞术和荧光测定法，观察3-NPA预处理对缺血2h再灌注24h脑梗死体积、神经细胞凋亡和caspase-3活性的影响。结果 缺血2h再灌注24h，3-NPA预处理组脑梗死体积减小22．3％，caspase-3活性降低13．9％，TUNEL阳性细胞数和细胞凋亡百分率分别减少47．0％和43．9％，与对照组比较差异有显著性。结论 3-NPA预处理可以诱导脑缺血耐受，降低caspase-3活性，抑制神经细胞凋亡可能是其机制之一。     

化学预处理的研究进展

化学预处理是继缺血预处理之后，近几年来出现的有关组织保护研究领域的新兴课题。文章简要介绍了化学预处理的概念和发展过程，重点论述了化学预处理机制的研究进展。     

N-ω-硝基-L-精氨酸海马注射对大鼠学习、记忆能力的影响及尼莫地平的作用

生理剂量的ＮＯ对神经系统的活动至关重要 ,突触传递的海马长时程增强 (ｌｏｎｇ ｔｅｒｍｐｏｔｅｎｔｉａｔｉｏｎ ,ＬＴＰ)可能就是由ＮＯ介导的〔1〕。ＮＯ的生成有赖于神经元中Ｃａ2 浓度 ,而ＮＯ反过来又可以调节细胞内Ｃａ2 水平。钙通道阻滞剂尼莫地平 (ｎｉｍｏｄｉｐｉｎｅ ,Ｎｉｍ)对痴呆患者的促智作用机制尚需进一步明确。　　一、材料与方法　　 1.实验动物 :2月龄Ｗｉｓｔａｒ大鼠 ,雄性 ,180～ 2 0 0ｇ ,由山东医科大学实验动物中心提供。模型组 9只 ,干预组 9只 ,生理盐水 (ＮＳ)对照组 10只 ,空白对照组 10只。　　试…     

雌激素对缺血性卒中的神经保护作用机制

大量临床研究和动物实验均表明 ,雌激素对卒中具有神经保护作用 ,其机制相当复杂 ,涉及多个方面。文章就近年来雌激素对缺血性卒中的神经保护作用机制的研究进行了综述     

天麻及其提取物对神经血管单元保护作用的研究进展

以往对神经保护的研究主要集中在神经元本身，认为只要阻断导致神经元坏死和凋亡机制中的一个或几个环节便可减轻神经元的进一步损伤。近年的研究发现，任何导致神经元损伤的原因如缺血缺氧、癫痫、感染、外伤、肿瘤、中毒、代谢障碍、退行性变或营养缺乏等都可能同时损伤胶质细胞和血管内皮细胞，后者的病理变化会加剧神经元的进一步损伤。因而提出神经血管单元（neurovaScularunit）的概念，认为在神经保护治疗的同时必须兼顾对神经胶质细胞和血管内皮的保护。     

年龄因素对瘢痕组织中神经构筑的影响

目的 探讨年龄对瘢痕组织神经构筑的影响。方法 26份瘢痕组织标本取自来我院行整形修复手术的26例患者,正常皮肤来自供皮区,利用HE染色,单克隆抗体免疫荧光法,免疫荧光显微镜下观察不同年龄瘢痕患者及正常皮肤中的神经丝蛋白（NFP）、蛋白基因产物（PGP9．5）在末梢神经组织中的标记情况。结果 创面愈合后1～20a,≤20岁和〉20岁患者瘢痕组织中的神经纤维密度差异无统计学意义;1～9a患者瘢痕组织中的神经纤维密度明显少于正常皮肤的密度,〉9a患者瘢痕组织中的神经纤维密度基本恢复到正常皮肤水平。结论 20岁前后两个年龄段患者瘢痕组织中神经纤维密度差异无统计学意义,瘢痕的年龄段差异可能是神经系统以外其他因素作用的结果。     

通过抑制神经细胞凋亡对帕金森病有神经保护作用的药物

帕金森病是常见的神经退行性疾病之一。本病的主要病理特征是黑质致密部多巴胺能神经元变性、缺失,而且这种细胞死亡形式是以凋亡为主。抗凋亡已成为治疗帕金森病的又一策略。本文主要对能通过抑制帕金森病黑质多巴胺能神经元凋亡而产生的神经保护作用的药物做以综述。     

The role of peroxynitrite in chemical preconditioning with 3-nitropropionic acid in rat hearts

OBJECTIVES: 3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, has been shown to protect against ischemic injury in the brain and in the heart via a preconditioning-like effect; however, the cellular mechanism is not known. The aim of the present study was to investigate if 3-NP pretreatment reduces infarct size and if altered metabolism of nitric oxide and reactive oxygen species are involved. METHODS: Hearts were assigned into 3 groups: 3 intermittent cycles of 5 min no-flow ischemia separated by 5 min aerobic perfusion protocol were used to induce ischemic preconditioning as a positive control; a time-matched non-preconditioning group served as control; and 3-NP (20 mg/kg, i.p.) was injected 3 h before the perfusion protocol to induce pharmacological preconditioning. Hearts from all groups were then subjected to 30 min global ischemia followed by 120 min reperfusion. RESULTS: Infarct size and lactate dehydrogenase release were significantly reduced after ischemia/reperfusion. While cardiac nitric oxide (NO) was increased, superoxide formation, nitrotyrosine level, and cardiac NADH oxidase and xanthine oxidase (XO) activities were markedly reduced by 3-NP administration. Cardiac activities of NO synthase and superoxide dismutase were not changed by 3-NP. CONCLUSION: This is the first demonstration in the rat myocardium that 3-NP induces pharmacological preconditioning, thereby limiting infarct size, and that this effect is associated with increased NO bioavailability and reduced peroxynitrite formation due to inhibition of superoxide formation by XO and NADH oxidase.     

维生素E对大剂量维生素D3所致心肌损伤的保护作用

本实验以大剂量维生素Ｄ３作为条件因素建立动物心肌损伤模型，观察维生素Ｅ对心肌损伤的保护作用。结果，大剂量维生素Ｄ３能降低大鼠心肌呼吸酶（细胞色素氧化酶和琥珀酸脱氢酶）活性。明显降低血清维生素Ｅ含量，使大鼠血清、心肌、肝脏组织谷胱甘肽过氧化物酶（ＧＳＨ—Ｐｘ）活性降低，脂质过氧化物（ＭＤＡ）含量升高。而预先给维生素Ｅ，能明显提高大鼠血清维生素Ｅ含量，提高呼吸酶活性．增强大鼠血清。心肌、肝脏组织ＧＳＨ—Ｐｘ活性，降低脂质过氧化物值。使大鼠心肌坏死面积明显减少。     

3-硝基丙酸对缺血预处理后脑组织caspase-3表达的影响

目的探讨脑缺血预处理后3-硝基丙酸(3-NPA)对脑组织中caspase-3表达的影响。方法72只健康雄性SD大鼠,随机分成3组:脑缺血组24只,脑缺血预处理组24只,脑缺血预处理后3-NPA干预组24只。各组再按缺血时间分为6h、1、2和4天4个时间点。结果在缺血后相同时间点,脑缺血预处理组与脑缺血组比较神经功能缺损评分明显降低(P<0.05),梗死体积明显缩小(P<0.05),海马区caspase-3阳性细胞表达明显降低(P<0.05);脑缺血预处理后3-NPA干预组神经功能缺损评分、梗死体积、caspase-3阳性细胞表达较脑缺血预处理组明显降低,差异有显著性意义(P<0.05)。各组在脑缺血后6h出现caspase-3阳性细胞表达,1～2天达到高峰后逐渐下降。结论缺血预处理后3-NPA干预可进一步加强神经保护作用。     

3-硝基丙酸对缺血预处理后脑组织caspase-3CasPase-3表达的影响

目的探讨脑缺血预处理后3硝基丙酸（3NPA）对脑组织中caspase-3表达的影响。方法72只健康雄性SD大鼠，随机分成3组：脑缺血组24只，脑缺血预处理组24只，脑缺血预处理后3NPA干预组24只。各组再按缺血时间分为6h、1、2和4天4个时间点。结果在缺血后相同时间点，脑缺血预处理组与脑缺血组比较神经功能缺损评分明显降低（P〈0.05），梗死体积明显缩小（P〈0.05），海马区caspase-3阳性细胞表达明显降低（P〈0.05）;脑缺血预处理后3NPA干预组神经功能缺损评分、梗死体积、caspase-3阳性细胞表达较脑缺血预处理组明显降低，差异有显著性意义（P〈0.05）。各组在脑缺血后6h出现caspase3阳性细胞表达，1～2天达到高峰后逐渐下降。结论缺血预处理后3NPA干预可进一步加强神经保护作用。     

多普勒导丝对腺苷预处理保护冠状动脉微循环功能的评价

目的 :观察腺苷预处理 (APC)对缺血心肌相关冠状动脉 (冠脉 )微循环功能的保护作用。方法 :将建立心肌缺血模型的 12只中国小型家猪随机分为两组 :对照组 6只 ,仅制备心肌缺血模型 ;APC组 6只 ,制备模型前经冠脉注入腺苷 6mg。应用冠脉内多普勒导丝观察模型相关冠脉平均峰值流速 (APV)、舒张收缩流速比值(DSVR)、冠脉血流储备 (CFR)的变化。结果 :两组模型冠脉狭窄后 10min时APV、DSVR、CFR均较基础状态时明显下降 (P <0 .0 5或 <0 .0 1) ,APV、DSVR在随后实验过程中无明显变化 ;30min时两组CFR均有进一步降低趋势 (P >0 .0 5 ) ;6 0min时对照组CFR较 30min时明显降低〔(0 .96± 0 .2 7)∶(1.74± 0 .4 9) ,P <0 .0 5〕 ,而APC组CFR与 30min时相比差异无统计学意义〔(1.6 4± 0 .30 )∶(1.79± 0 .38) ,P >0 .0 5〕 ;但 12 0min时APC组CFR较前明显下降〔(0 .94± 0 .31)∶(1.6 4± 0 .30 ) ,P <0 .0 1〕 ,与同时间点对照组相比差异无统计学意义。结论 :心肌缺血模型相关冠脉在急性重度狭窄 6 0min时发生微循环功能障碍 ,APC对冠脉微循环功能具有保护作用 ,但持续时间较短。     

The antiapoptotic effect of mesenchymal stem cell transplantation on ischemic myocardium is enhanced by anoxic preconditioning

BACKGROUND:

Cardiomyocyte apoptosis takes place at an early stage after myocardial infarction (MI). Therapy with mesenchymal stem cells (MSCs) is reported to reduce apoptosis.

OBJECTIVES:

To determine whether anoxic preconditioning (AP) could enhance the antiapoptotic effect of MSCs.

METHODS:

Cultured cardiomyocytes were treated with Dulbecco’s modified Eagle’s medium (as a control), MSCs or AP-MSCs, and were exposed to hypoxia/reoxygenation. Apoptotic cardiomyocytes were stained with Annexin V fluorescein isothiocyanate (BioVision, USA), visualized by fluorescence microscopy and analyzed by flow cytometry. In vivo, MI was produced in Sprague-Dawley rats by permanent ligation of the left anterior descending coronary artery and the left ventricles were randomly injected with Dulbecco’s modified Eagle’s medium, MSCs or AP-MSCs one week after MI. The cardiomyocyte apoptotic rate in peri-infarcted areas was assessed by terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate nick end labelling assay one week after transplantation. Cardiac function was assessed by echocardiography four weeks after transplantation. Infarct size was measured by hematoxylin and eosin staining one and four weeks after transplantation. The expression of Bcl-2, Bax protein and cleaved cysteine-aspartic acid protease-3 was analyzed by Western blot techniques.

RESULTS:

Cardiomyocyte apoptosis (both induced by hypoxia/reoxygenation and MI) was significantly reduced by treating with MSCs and AP-MSCs, the Bcl-2 to Bax protein ratio was increased and cleaved cysteine-aspartic acid protease-3 was decreased. AP-MSCs were superior to MSCs.

CONCLUSIONS:

MSCs protected the infarcted heart by preventing cardiomyocyte apoptosis and AP enhanced the cardioprotective effects of MSCs.     

Dilazep dihydrochloride prolongs the infarct size-limiting effect of ischemic preconditioning in rabbits

Recent studies have indicated the key role of adenosine receptor activation as a trigger for ischemic preconditioning (PC). Hence, the augmentation of endogenous adenosine may potentiate the cardioprotective effects of PC. In this study, we aimed to test the hypothesis that dilazep dihydrochloride, an adenosine transport inhibitor, potentiates the PC effect. Protocol 1: Infarcts were produced in open-chest anesthetized rabbits by 30-min occlusion of a coronary artery and 2 days' reperfusion. PC was elicited by a preceding 5-min occlusion and either 5, 40, or 120 min of reperfusion. PC with the 5-min reperfusion markedly limited the infarct size after the 30-min ischemia (infarct size to area at risk (IS): 10% ± 3% vs 41% ± 3%, P < 0.05). PC was not protective when the reperfusion periods were 40 or 120 min (IS: 47% ± 5% and 44% ± 3%, P = not significant (NS) vs control, respectively). However, concomitant treatment with dilazep (0.2 mg/kg) preserved the PC effect in the 40-min reperfusion group (18% ± 5%, P < 0.05 vs control) but not in the 120-min reperfusion group (43% ± 4%, P = NS vs control). Protocol 2: Infarct was produced in a similar rabbit model by either a 45- or 50-min occlusion of a coronary artery and 2 days of reperfusion. PC was elicited by a preceding 5-min occlusion and a 5-min reperfusion. PC was protective in the 45-min occlusion group (30% ± 7% vs 67% ± 3%, P < 0.05) but not in the 50-min occlusion group (74% ± 4% vs 79% ± 5%, P = NS). Treatment with dilazep (0.2 mg/kg) failed to retrieve protection in this preconditioned group (77% ± 6%, P = NS vs 50-min occlusion group without PC). Thus, dilazep prolonged the infarct size-limiting effect of PC, but failed to retrieve protection in the group with a longer sustained ischemia. Received: September 6, 2000 / Accepted: March 9, 2001     

Evidence that the acute phase of ischemic preconditioning does not require signaling by the A2B adenosine receptor

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A_2BAR gene knock-out (A_2BKO)/β-galactosidase reporter gene knock-in mice and the A_2BAR antagonist ATL-801 to investigate the potential involvement of the A_2BAR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A_2BKO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A_2BKO hearts by ~ 30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A_2BKO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A_2BKO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A_2BAR agonist BAY 60-6583 (~ 25% reduction at a dose of 1.0 mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A_2BAR. We present the idea that the A_2BAR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.     

Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.     

Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA

We have shown that the cellular process of macroautophagy plays a protective role in HL-1 cardiomyocytes subjected to simulated ischemia/reperfusion (sI/R) (Hamacher-Brady et al. in J Biol Chem 281(40):29776–29787). Since the nucleoside adenosine has been shown to mimic both early and late phase ischemic preconditioning, a potent cardioprotective phenomenon, the purpose of this study was to determine the effect of adenosine on autophagosome formation. Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles, and can be monitored by imaging the incorporation of microtubule-associated light chain 3 (LC3) fused to a fluorescent protein (GFP or mCherry) into nascent autophagosomes. We investigated the effect of adenosine receptor agonists on autophagy and cell survival following sI/R in GFP-LC3 infected HL-1 cells and neonatal rat cardiomyocytes. The A₁ adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) (100 nM) caused an increase in the number of autophagosomes within 10 min of treatment; the effect persisted for at least 300 min. A significant inhibition of autophagy and loss of protection against sI/R measured by release of lactate dehydrogenase (LDH), was demonstrated in CCPA-pretreated cells treated with an A₁ receptor antagonist, a phospholipase C inhibitor, or an intracellular Ca(+2) chelator. To determine whether autophagy was required for the protective effect of CCPA, autophagy was blocked with a dominant negative inhibitor (Atg5^K130R) delivered by transient transfection (in HL-1 cells) or protein transduction (in adult rat cardiomyocytes). CCPA attenuated LDH release after sI/R, but protection was lost when autophagy was blocked. To assess autophagy in vivo, transgenic mice expressing the red fluorescent autophagy marker mCherry-LC3 under the control of the alpha myosin heavy chain promoter were treated with CCPA 1 mg/kg i.p. Fluorescence microscopy of cryosections taken from the left ventricle 30 min after CCPA injection revealed a large increase in the number of mCherry-LC3-labeled structures, indicating the induction of autophagy by CCPA in vivo. Taken together, these results indicate that autophagy plays an important role in mediating the cardioprotective effects conferred by adenosine pretreatment.