干预Rac1活性在大鼠心脏骤停全脑缺血模型中的脑保护作用

发布时间：2018-01-14 06:09

本文关键词：干预Rac1活性在大鼠心脏骤停全脑缺血模型中的脑保护作用　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：由各种疾病(脑卒中、微血栓、脑血管痉挛和硬化、脑血液动力学改变、颈部动脉疾病或椎动脉受压等)或手术(严重颅脑外伤手术、控制性降压、颅内动脉瘤夹闭术、冠状动脉旁路移植术以及颈动脉血管移植术、蛛网膜下腔出血等)所介导的全部或局部的脑组织短暂缺血,缺血脑组织恢复血流灌注后,脑组织损伤反而加重称为脑缺血再灌注损伤(ischemia-reperfusion injury, I/R)。神经细胞对缺血性损害非常敏感,长时间的缺血打击可导致神经细胞大量死亡,难以再生,从而留下许多严重甚至是不可逆的后遗症。因此,如何实现围术期脑保护一直是临床麻醉医师追求的目标。研究某种能够调动机体的内源性保护机制,提高神经细胞对缺血性损害的抵抗力,减少严重缺血导致的损伤,保证再灌注后神经细胞的正常生理功能,是缺血性脑病治疗上的一个重要策略。探求脑缺血再灌注损伤的发病机制、寻找可减轻或预防脑缺血再灌注损伤的方法或药物,已成为近年来神经科学领域广大科研工作者致力的研究目标。全脑缺血后活性氧尤其是氧自由基所引起的连锁反应是神经元受损的核心病理环节。Rac1蛋白被认为是还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶的生物调控开关,当有外源性的刺激因素存在时,Rac1与GDP脱离,并与三磷酸鸟苷GTP相结合而活化,随后激活NADPH氧化酶从而产生活性氧簇。目前一些脑缺血再灌注研究发现在各种手段作用下降低Rac1活性将避免产生过多的活性氧参与脑缺血后神经元的死亡或凋亡,但是针对Rac1在全脑缺血中的作用及其信号转导的调节机制方面的研究仍然较少,其信号转导通路在脑损伤后是否受到其他因素的影响目前尚未明确,作为活性氧产生通路上的“开关分子’Rac1与线粒体的抗氧化酶系统是否有相关调节机制参与脑缺血再灌注损伤还需进一步探索。本实验首先利用食道电极建立心室停搏CA(cardiac arrest)全脑缺血GCI(global cerebral ischemia)模型,并在全脑缺血15min前经侧脑室注射Rac1特异抑制剂NSC23766(50μg),观察记录大鼠9天内生存情况,通过Niss1染色评价大鼠海马CA1区在脑缺血再灌注后神经元形态学改变,观察其中存活神经元密度。TUNEL染色检测48h海马CA1区的神经元细胞的凋亡情况,观察侧脑室注射Rac1特异抑制剂NSC23766是否可减轻GCI后海马神经元损伤,证明该模型可以一定程度模拟临床各种原因导致的心脏停搏引发的严重全脑缺血损害；NADPH氧化酶“开关分子’Rac1与全脑缺血再灌损伤有关。第二部分以此模型为基础研究抑制Rac1活性对大鼠CA后脑神经功能和氧化应激的影响,通过侧脑室注射Rac1活性抑制剂NSC23766,于CA/GCI后30min、3h、6h、1d、3d时取海马CA1区组织检测Rac1总量及活化蛋白含量,从WB水平验证侧脑室注射NSC23766对Rac1活性的影响,通过改良NSS评分和Morris水迷宫检测空间学习与记忆能力的等神经功能改变,检测超氧化物歧化酶(SOD)、丙二醛(MDA)等指标来反映神经元的氧化应激水平,结果证明抑制Rac1活性对全脑缺血再灌损伤后大鼠空间学习及记忆能力有保护作用,同时抑制Rac1活性可降低神经元氧化应激水平,提示抑制Rac1活性所产生的神经保护作用与降低神经元氧化应激水平有关。第三部分则于CA/GCI后再灌注6h、1d、3d、5d各时间点检测大鼠海马CA1区Trx2, Prx3的蛋白时间表达分布水平,研究线粒体抗氧化酶Trx2, Prx3在Rac1抑制剂处理后大鼠脑缺血再灌注损伤中的变化,探讨机体清除氧化产物的还原能力特别是线粒体抗氧化酶系统是否被辅助激活,关注抑制Rac1活性所产生的脑保护作用是否与激活、增强神经元细胞的抗氧化酶系统清除活性氧能力有关。我们进一步研究线粒体抗氧化酶在全脑缺血再灌注的保护机制,期望为防治全脑I/R损伤提供新的治疗靶点。实验发现：①抑制Rac1活性可明显减少缺血再灌注所导致的迟发性神经元死亡及锥体神经细胞凋亡,并可以使大鼠的空间学习和记忆能力的减退得到明显改善,起到在全缺血再灌注损伤中的神经保护作用。②使用Rac1抑制剂NSC23766降低Racl活性对大鼠再灌注损伤的脑保护作用与改善氧化应激水平及氧化应激的脑组织相关蛋白分子相关联。③Racl活性抑制剂处理后减轻大鼠脑缺血再灌注损伤降低组织氧化应激水平的机制可能与脑组织线粒体抗氧化酶Trx2.Prx3表达无关。本研究为进一步阐明脑缺血再灌注损伤的机制奠定理论基础,也为Rac1与线粒体抗氧化酶Trx2, Prx3在缺血性脑损伤的重要作用提供理论依据和治疗策略,同时尝试为治疗临床缺血性脑病提供一条新的思路。第一部分抑制Racl活性对大鼠经食道致颤脑缺血模型的神经元保护作用摘要：目的：建立SD大鼠经食道电刺激心脏骤停全脑缺血再灌注模型,研究侧脑室注射NSC23766抑制Racl活性在该模型中的神经元保护作用。方法：经食道插入调搏电极至心脏水平,用恒定电流诱发心脏骤停,无干预观察6min后进行心肺复苏。选择雄性SD大鼠(250-300g),随机分为四组：Sham组,CA组,NSC组(全脑缺血15min前经侧脑室置管注射Rac1特异抑制剂NSC23766), Vehicle组。记录各组大鼠9d内生存情况,于I/R后2d检测各组大鼠脑水肿情况,大鼠海马CA1区行TUNEL染色记录凋亡阳性细胞数,I/R后9d取大鼠海马CA1区行Nissl染色,观察存活神经元密度。结果：大鼠心脏骤停后全脑缺血NSC23766治疗组与CA模型组相比生存率显著提高(P0.05)。NSC组与CA组相比脑水含量减少,海马CA1区存活神经细胞数目增多(P0.05),迟发性神经元死亡明显减少(P0.05)。结论：经食道电刺激心脏骤停全脑缺血模型可模拟临床心脏骤停后脑损害；抑制Rac1活性可明显减少缺血再灌注所导致的迟发性神经元死亡及锥体神经细胞凋亡。第二部分抑制Racl活性对大鼠CA后脑神经功能和氧化应激的影响摘要：目的：探讨抑制Rac1活性对大鼠心脏骤停全脑缺血后脑神经保护作用与氧化应激的关系。方法：经食道插入调搏电极至心脏水平,用恒定电流诱发心脏骤停,无干预观察6min后进行心肺复苏制作全脑缺血再灌注模型。选择雄性SD大鼠(250-300g),随机分为四组：Sham组,CA组,NSC组,Vehicle组(CA前15min经侧脑室置管注射NSC23766)。于再灌注后6h、1d、2d及4d时行NSS评分；再灌注第2d各组检测超氧化物歧化酶(SOD)、丙二醛(MDA),第7d行Morris水迷宫实验。结果：缺血再灌注后6小时大鼠海马CA1区Racl活性明显高于假手术组(P0.05)。同CA组相比,NSC组缺血再灌注后6h大鼠海马CA1区Racl活性显著降低,改良NSS评分各时间点都降低,Morris水迷宫实验中缺血再灌后第7天和第8天搜索安全岛平台潜伏期、运动轨迹有明显改善,空间探索试验时NSC组第2象限停留时间百分比和穿越原平台的次数明显增加(P0.05)。氧化应激检测结果显示,缺血再灌发生时抗氧化物质SOD降低而脂质氧化标志物MDA升高,NSC组与CA组相比水平升高,MDA水平降低(P0.05)。结论：抑制Rac1活性在大鼠经食道电刺激心脏骤停全脑缺血模型中可以通过降低氧化应激水平,改善大脑缺血再灌注的神经功能损伤,发挥其在全脑I/R损伤中的神经保护作用。第三部分线粒体抗氧化酶Trx2, Prx3在大鼠脑缺血再灌注损伤中的变化摘要：目的：通过评价线粒体抗氧化酶Trx2, Prx3在Rac1抑制剂处理后大鼠脑缺血再灌注损伤中的蛋白表达变化,探讨线粒体抗氧化酶在大鼠脑缺血再灌注损伤的可能保护机制。方法：经食道插入调搏电极至心脏水平,用恒定电流诱发心脏骤停CA,无干预观察6min后进行心肺复苏制作全脑I/R模型。选择雄性SD大鼠(250-300g),随机分为四组：Sham组,CA组,NSC组(全脑缺血15min前经侧脑室注射Rac1特异抑制剂NSC23766), Vehicle组。于再灌注后6h、1d、3d、5d取海马CA1区组织行Western blot检测硫氧还蛋白酶Trx2及线粒体过氧化氢酶Prx3的表达。结果：CA组Trx2与Prx3在缺血再灌注1d后表达稳定,两种蛋白与CA组6h时表达相比均有差异(P0.05),与3d表达无统计学差异；NSC组与CA组比较,Trx2、Prx3蛋白表达差异无统计学意义。结论：Rac1抑制剂处理后减轻大鼠脑缺血再灌注损伤的机制可能与脑组织线粒体抗氧化酶Trx2、Prx3表达调节无关。
[Abstract]:By a variety of diseases (stroke, micro thrombosis, cerebral vasospasm and cerebral hemodynamic changes, atherosclerosis, carotid artery disease or vertebral artery compression) or surgery (severe craniocerebral trauma surgery, controlled hypotension, intracranial aneurysm surgery, coronary artery bypass grafting and carotid artery transplantation, subarachnoid hemorrhage etc.) mediated by all or part of the brain ischemia, ischemia reperfusion, brain damage aggravate called cerebral ischemia reperfusion injury (ischemia-reperfusion, injury, I/R).
Nerve cells are very sensitive to ischemic damage, long time ischemia can lead to a large number of dead neural cells to regenerate, leaving many serious and even irreversible sequelae. Therefore, how to realize the perioperative brain protection has been a clinical anesthesiologists in the pursuit of the goal. Some research can arouse the endogenous protective mechanism of organism, improve resistance force of nerve cells to ischemic injury, reduce the severity of ischemic damage, ensure the normal physiological function of nerve cells after reperfusion, is an important strategy for the treatment of ischemic encephalopathy. Look for cerebral ischemia reperfusion injury, for reducing or preventing cerebral ischemia reperfusion injury or drug, has been become the research target in recent years the field of neuroscience researchers working.
After cerebral ischemia especially active oxygen oxygen free radical chain reaction is caused by the damaged neurons of the core pathology link.Rac1 protein is considered to be the biological control switch also reduced nicotinamide adenine dinucleotide phosphate oxidase, when the presence of exogenous stimuli, Rac1 and GDP from, and combined with three GMP and GTP activation and subsequent activation of NADPH oxidase to generate reactive oxygen species. Some studies found that cerebral ischemia reperfusion decreased Rac1 activity in a variety of means under the action to avoid excessive generation of reactive oxygen species after cerebral ischemia and neuronal cell death or apoptosis, but the study on regulation mechanism of Rac1 in the global cerebral ischemia and signal transduction the still small, whether the signal transduction pathway under the influence of other factors is not clear in the brain after injury, as the production of reactive oxygen species on the pathway" It is necessary to further explore whether the switch molecule 'Rac1 and the mitochondrial antioxidant enzyme system have a related regulatory mechanism to participate in cerebral ischemia reperfusion injury.
This experiment firstly established esophageal electrode asystole (cardiac CA arrest GCI (global) cerebral ischemia model, and cerebral ischemia) in cerebral ischemia before 15min after intracerebroventricular injection of Rac1 inhibitor NSC23766 (50 g), observed and recorded the rats within 9 days of survival, through Niss1 staining in hippocampal CA1 evaluation rat morphologic changes of neurons in cerebral ischemia reperfusion, apoptosis of neurons was observed with.TUNEL staining density of neurons in hippocampal CA1 region of 48h detection, observation of intracerebroventricular injection of Rac1 inhibitor NSC23766 can reduce the injury of hippocampal neurons after GCI, proved that the model can serious cerebral ischemia damage to a certain extent the cause of clinical simulation all kinds of cardiac arrest caused by NADPH oxidase; "molecular switch" Rac1 with global cerebral ischemia reperfusion injury.
The second part based on the model of inhibitory effect of Rac1 activity on CA of cerebral nerve function and oxidative stress in rats, by intracerebroventricular injection of Rac1 inhibitor NSC23766, after CA/GCI 30min, 3h, 6h, 1D, 3D and CA1 region of the hippocampus tissues Rac1 and activation of total protein content, from the side effects of WB level verification intracerebroventricular injection of NSC23766 on the activity of Rac1, the modified NSS score and Morris water maze spatial learning and memory ability of the neural function, detection of superoxide dismutase (SOD), malondialdehyde (MDA) and other indicators to reflect the level of oxidative stress in neurons, results show that the inhibition of Rac1 activity has a protective effect on the brain ischemia reperfusion injury after rat spatial learning and memory ability, and inhibit the activity of Rac1 can reduce the level of oxidative stress in neurons, suggesting that the neuroprotective effect produced by inhibiting the activity of Rac1 and reduce the oxidation of neurons The level of stress is related.
The third part is on the CA/GCI after reperfusion for 6h, 1D, 3D, 5D at the time of detection of Trx2 CA1 of rat hippocampus, the expression level of Prx3 protein distribution time of mitochondrial antioxidant enzymes Trx2, Prx3 in Rac1 treated rats cerebral ischemia reperfusion injury of change, reduction ability of machine body to remove oxidation the product is especially mitochondrial antioxidant system is whether the brain protective effect of assisted activation, attention to inhibit the activity of Rac1 produced by the activation of neurons, enhance the antioxidant system of reactive oxygen scavenging capacity. We further study the protective mechanism of antioxidant enzymes in mitochondria in cerebral ischemia reperfusion, expect to provide new therapeutic targets for prevention and treatment of cerebral I/R injury.
The experiment showed that: the inhibition of Rac1 activity can significantly reduce ischemia reperfusion caused by delayed neuronal death and pyramidal neuronal apoptosis, and may cause the loss of spatial learning and memory ability of rats was obviously improved, play in the ischemia reperfusion injury in the neuroprotective effect. The use of Rac1 inhibitors reduce NSC23766 the molecular brain oxidative stress and oxidative stress and improve the protection of the activity of Racl on reperfusion injury of rat brain tissue related protein associated with mitochondrial processing. The inhibitor of Racl activity after cerebral ischemia reperfusion injury in rats reduced oxidative stress in low level of organization and possible mechanism of antioxidase in brain Trx2.Prx3 expression.
This study laid a theoretical foundation for further elucidation of the mechanisms of cerebral ischemia reperfusion injury, as well as Rac1 and mitochondrial antioxidant enzymes Trx2, Prx3 and provide a theoretical basis and treatment strategies play an important role in ischemic brain injury, and try to provide a new idea for clinical treatment of ischemic encephalopathy.
The first part inhibits the protective effect of Racl activity on the rat model of cerebral ischemia induced by the esophagus
Abstract: Objective: to establish SD rat transesophageal electrical stimulation of cardiac arrest cerebral ischemia reperfusion model, neuron protective effect of intracerebroventricular injection of NSC23766 inhibited the activity of Racl in this model. Methods: the transesophageal pacing electrode insertion to the level of the heart, with a constant current induced cardiac arrest, without intervention after 6min CPR. Select male SD rats (250-300g), were randomly divided into four groups: Sham group, CA group, NSC group (global cerebral ischemia before 15min by intraventricular catheter injection of a specific inhibitor of Rac1 NSC23766), Vehicle group. The rats were recorded. 9D internal storage, I/R 2D to detect brain edema of rats, CA1 of rat hippocampus by TUNEL staining to record the number of apoptotic cells, I/R 9D CA1 of rat hippocampus by Nissl staining to observe the survival neuron density. Results: the rat cardiac arrest after cerebral ischemia NSC23766 treatment group and CA model group. The ratio of survival increased significantly (P0.05). Compared with the CA group, the.NSC content of the brain decreased significantly, and the number of viable neurons in the CA1 area increased (P0.05), and the delayed neuronal death was significantly reduced (P0.05).
Conclusion: the esophageal electrical stimulation model of global cerebral ischemia can simulate brain damage after cardiac arrest. Inhibition of Rac1 activity can significantly reduce delayed neuronal death and pyramidal nerve cell apoptosis induced by ischemia-reperfusion.
The second part inhibits the effect of Racl activity on the nerve function and oxidative stress in the rat brain after CA
Abstract: Objective: To investigate the inhibition of Rac1 activity on cardiac arrest in rats after cerebral ischemia and neuroprotective effects of oxidative stress. Methods: the transesophageal pacing electrode insertion to the level of the heart, with a constant current induced cardiac arrest, no intervention on 6min after cardiopulmonary resuscitation for cerebral ischemia reperfusion model. Male SD rats (250-300g), were randomly divided into four groups: Sham group, CA group, NSC group, Vehicle group (CA 15min before the lateral ventricle catheter injection of NSC23766). After reperfusion, 6h, 1D, 2D and 4D were NSS score; reperfusion in 2D were detected by superoxide dismutase (SOD), malondialdehyde (MDA), the 7d Morris water maze test. Results: 6 hours after ischemia reperfusion Racl in rat hippocampal CA1 region was significantly higher than that of sham operation group (P0.05). Compared with the CA group, NSC group, Racl after ischemia reperfusion in hippocampal CA1 area of rats significantly decreased the activity of 6h. The modified NSS score Time points are reduced in Morris water maze after ischemia reperfusion for seventh days and eighth days of searching safety island platform latency, the trajectory is obviously improved, the number of space exploration test group NSC second quadrant time and percentage of crossing the original platform significantly increased (P0.05). Oxidative stress test results showed that the occurrence of ischemia reperfusion when the antioxidant SOD decreased lipid peroxidation markers MDA increased in NSC group compared with CA group increased the level of MDA decreased (P0.05). Conclusion: the inhibition of Rac1 activity in rat cardiac arrest by transesophageal electrical stimulation of cerebral ischemia model can reduce oxidative stress level, nerve function improve brain ischemia reperfusion injury in the play, the whole brain I/R injury in neuroprotective effect.
The changes of mitochondrial antioxidant enzyme Trx2 and Prx3 in the third part of cerebral ischemia reperfusion injury in rats
Abstract: Objective: To evaluate the mitochondrial antioxidant enzymes Trx2, Prx3 in Rac1 inhibitor treatment after cerebral ischemia reperfusion injury in rats. The protein expression changes of mitochondrial antioxidant enzymes in rat cerebral ischemia reperfusion injury of the possible protective mechanism. Methods: the transesophageal pacing electrode insertion to the level of the heart, with a constant current induced heart arrest CA, without intervention on 6min after cardiopulmonary resuscitation made the whole brain I/R model. Select male SD rats (250-300g), were randomly divided into four groups: Sham group, CA group, NSC group (global cerebral ischemia 15min after intracerebroventricular injection of Rac1 inhibitor NSC23766), Vehicle group after reperfusion. 6h, 1D, 3D, 5D in CA1 area of hippocampus tissue Western blot detection of thioredoxin reductase and mitochondrial Trx2 catalase Prx3 expression. Results: the expression of Trx2 and Prx3 in stable CA group 1D after reperfusion, the expression of two kinds of protein with CA group 6H The differences were compared (P0.05), there was no significant difference in the expression of 3D and NSC; compared with group CA and group Trx2, Prx3 expression had no statistical difference. Conclusion: mitochondrial Rac1 inhibitor treatment after cerebral ischemia reperfusion injury in rats and possible mechanisms of brain tissue antioxidant enzymes Trx2, Prx3 expression.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R-332;R743

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