基于Cofilin介导细胞凋亡探讨电针促进脑缺血大鼠认知功能恢复的作用机制

发布时间：2018-04-26 21:09

  本文选题：丝切蛋白 + 电针　； 参考：《福建中医药大学》2017年博士论文

【摘要】：缺血性卒中已成为世界各地长期神经功能障碍和死亡的主要原因,约占所有卒中的80%。尽管已经有几十年的深入研究,但能够针对性地治疗缺血性卒中的措施仍然有限。脑缺血的主要致病机制包含谷氨酸介导的兴奋性毒性,钙超载,炎症反应,血脑屏障破坏和氧化应激等。这些病理因素或者环节相互影响,互为因果,进而引起神经细胞凋亡与坏死,最终导致卒中后认知功能障碍。广泛认为半暗带中的细胞凋亡是可逆的过程。细胞凋亡的调控,在保护神经元和改善认知的过程中发挥着重要的作用。因此,探讨神经细胞凋亡的分子机制,寻找有效的治疗方法减少脑损伤,已成为脑血管研究领域的重点和热点。丝切蛋白(Cofilin)是肌动蛋白结合蛋白,受LIMK介导的磷酸化而失活。过量谷氨酸,ATP耗竭或氧化应激处理原代培养神经元,均发现Cofilinrod结构的形成。产生这种棒状结构的神经元在突触结构、突触转运和长时程突触可塑性上都受到了一定程度损伤。Rod的形成主要是由于去磷酸化的Cofilin含量增加引起的,并且仅去磷酸化的Cofilin牵涉线粒体转位。Cofilin线粒体转位在细胞凋亡程序的启动中起到关键作用。此外,引起Rod形成的病理性刺激,均是缺血性卒中的重要病理机制。由此提示,Cofilin及其介导的Rod结构与学习记忆等认知功能密切相关,同时参与缺血性卒中细胞凋亡过程中。目前,电针疗法已成为脑卒中后康复治疗的一种常见方法。督脉经穴"百会"和"神庭"具有益气升阳、填髓充脑、醒脑开窍之功效,常被用于治疗神经和精神疾病,如中风,头痛和头晕等。临床和基础研究均显示,电针"百会"和"神庭"穴有利于保护卒中患者和动物模型的缺血性损伤。本课题组的临床研究显示,针刺"百会"、"神庭"穴在认知康复训练基础上可显著提高脑卒中患者MMSE、MoCA和FIM等认知相关量表的评分。同时,我们以往实验也发现电针"百会"、"神庭"穴能够通过减少脑缺血损伤大鼠的神经细胞凋亡,显著改善其认知功能障碍。然而,电针治疗缺血性卒中的分子机制尚不明确。目的:(1)阐明Cofilin在缺血性卒中后细胞凋亡中的潜在调控作用,为将Cofilin作为脑卒中新的治疗靶点提供实验依据;(2)探讨电针"百会"、"神庭"穴促进脑缺血大鼠认知功能恢复的可能分子机制,为中医康复临床实践提供一定的理论依据。方法:第一部分Cofilin在脑缺血大鼠皮质梗死区细胞凋亡中的作用(1)本研究利用ATP耗竭诱导原代海马神经元,建立体外缺血/缺氧损伤模型,明确Cofilin rod形成和细胞凋亡。(2)同时,以大脑中动脉闭塞法制备缺血性卒中的实验动物模型,通过TTC和NeuN染色来评估MCAO大鼠脑梗死的发展阶段。(3)应用免疫组化和TUNEL染色,观察不同时间点(2h,8h,24h和7d)MCAO大鼠脑皮质梗死区Cofilin rod生成和神经细胞凋亡的情况。(4)为进一步证实去磷酸化Cofilin在脑缺血中的作用,我们利用Limk1过表达抑制Cofilin的活化,观察MCAO大鼠缺血半暗带病毒感染区域Rod形成和Cofilin介导细胞凋亡的情况。第二部分电针"百会"、"神庭"穴调控Cofilin线粒体转位减少皮质缺血区细胞凋亡的机制研究(1)分别于脑缺血术后5min和6h,对MCAO大鼠进行电针干预"百会"、"神庭"穴。(2)通过条件恐惧记忆以及神经功能缺损得分和脑梗死体积,评价电针"百会"、"神庭"对脑缺血大鼠的神经保护作用。(3)采用Nissl染色和荧光染色以观察脑缺血组织损伤、Cofilin rod形成、细胞凋亡和神经元丢失情况。(4)并通过Western blot检测缺血半暗带Caspase-3的活性,及其胞浆和线粒体中Cofilin和p-cofilin的表达水平。结果:第一部分(1)ATP耗竭可诱导原代海马神经元Cofilin rod形成以及细胞凋亡。(2)TTC和NeuN染色显示梗死体积从2h增加到24h,然后在MCAO后24h到7d稳定。(3)Rod结构在梗死核心区2h和半暗带8h开始生成,24h达到峰值,然后在MCAO后7d减少。而逐步增多的Rod结构可能会逐渐诱导脑卒中后梗死皮层MAP2降解和细胞凋亡。(4)Limk1过表达抑制Cofilin的活化,可显著降低MCAO大鼠缺血区半暗带Rod的形成和MAP2降解,并减少Cofilin介导的细胞凋亡,进一步证实了Cofilin在脑缺血中的作用。第二部分(1)急性期电针"百会"、"神庭"穴治疗MCAO大鼠可以显著减小脑梗死体积,并改善脑损伤引起的神经功能缺损症状和认知功能障碍。(2)Western blot分析表明,电针这两个穴位还可以明显抑制Cofilin的线粒体转位和Caspase-3切割。(3)此外,免疫荧光双标结果提示,电针"百会"、"神庭"穴治疗不仅可以部分抑制Cofilin rod形成和MAP2降解,而且还显著减少缺血性卒中诱导的脑组织损伤、细胞凋亡和神经元丢失。结论:(1)体内和体外缺血可诱导Cofilin rod形成和神经元细胞凋亡,而Limk1过表达抑制Cofilin过度激活,可显著减少脑缺血后皮质半暗带Rod形成、MAP2降解和Cofilin线粒体转位介导的细胞凋亡,由此可知,Cofilin对缺血性卒中后Rod形成和细胞凋亡具有重要作用,可作为缺血性卒中潜在的治疗靶点。(2)急性期电针"百会"、"神庭"穴可减少Cofilin rod形成和MAP2降解,抑制Cofilin的线粒体转位,减轻缺血性卒中的脑损伤和细胞凋亡,从而促进认知功能恢复,这可能是电针疗法的新机制。
[Abstract]:Ischemic stroke has become a major cause of long-term neurological dysfunction and death in all parts of the world. The 80%., which accounts for all strokes, has been studied for decades, but the measures to treat ischemic stroke are still limited. The main pathogenesis of cerebral ischemia includes glutamate mediated excitotoxicity, calcium overload, and inflammation. These pathological factors or links interact with each other, cause cause and effect, cause apoptosis and necrosis of nerve cells, and eventually lead to cognitive impairment after stroke. It is widely considered that apoptosis in the semi dark zone is a reversible process. Apoptosis regulation, in protecting neurons and improving cognition It has played an important role in the process. Therefore, to explore the molecular mechanism of neuronal apoptosis and to find an effective treatment method to reduce brain damage has become the focus and hot spot in the field of cerebral vascular research. Cofilin is actin binding protein, LIMK mediated phosphorylation and inactivation. Excessive glutamate, ATP depletion or oxidative stress The formation of the Cofilinrod structure is found in the primary cultured neurons. The neurons that produce this rod like structure have been damaged to a certain extent in the synaptic structure, the synaptic transport and the long term synaptic plasticity. The formation of the.Rod is mainly due to the increase of the Cofilin content of dephosphorylation, and the Cofilin involvement line is only dephosphorylated. Mitochondrial translocation of granular.Cofilin plays a key role in the initiation of apoptosis procedures. In addition, the pathological stimulation of Rod formation is an important pathological mechanism of ischemic stroke. Thus, it is suggested that Cofilin and its mediated Rod structure are closely related to cognitive functions such as learning and memory, and are involved in the apoptosis of ischemic stroke cells. At present, electroacupuncture has become a common method of rehabilitation after stroke. The meridian meridian "Baihui" and "Shen Ting" have the effect of replenishing qi and Yang, filling the brain into the brain, and activating the brain, often used in the treatment of nerve and mental illness, such as stroke, headache, dizziness and so on. Clinical and basic studies all show the electric acupuncture "hundred meeting" and "Shen Ting" points. It is beneficial to protect the ischemic injury of stroke patients and animal models. The clinical study of this group has shown that acupuncture of "Baihui" and "Shen Ting" point can significantly improve the score of cognitive related scales of stroke patients, such as MMSE, MoCA and FIM on the basis of cognitive rehabilitation training. Reduce the apoptosis of neurons in rats with cerebral ischemia and improve their cognitive impairment. However, the molecular mechanism of electroacupuncture in the treatment of ischemic stroke is not clear. Objective: (1) to clarify the potential regulation of Cofilin in cell apoptosis after ischemic stroke, and to provide experimental basis for Cofilin as a new target for stroke treatment; (2) To discuss the possible molecular mechanism of "Baihui" and "Shen Ting" point to promote the recovery of cognitive function in rats with cerebral ischemia, and provide some theoretical basis for the clinical practice of TCM rehabilitation. Method: the role of Cofilin in the apoptosis of cortical infarct area of cerebral ischemia rats (1) this study used the depletion of ATP to induce the primary hippocampal neurons to establish in vitro deficiency. Blood / hypoxia damage model, Cofilin rod formation and apoptosis. (2) at the same time, the experimental animal model of ischemic stroke was prepared by middle cerebral artery occlusion, and the development stage of cerebral infarction in MCAO rats was evaluated by TTC and NeuN staining. (3) immunohistochemistry and TUNEL staining were used to observe the cerebral cortex of MCAO rats at different time points (2h, 8h, 24h and 7D). The formation of Cofilin rod and the apoptosis of nerve cells in the dead tissue of the stem. (4) to further confirm the role of dephosphorylation of Cofilin in cerebral ischemia, we use Limk1 overexpression to inhibit the activation of Cofilin, and observe the situation of Rod formation and Cofilin mediated apoptosis in the ischemic penumbra virus infected region of MCAO rats. The second part of the electroacupuncture "hundred meetings", " The mechanism of "Shen Ting" regulating Cofilin mitochondrial transposition to reduce cell apoptosis in cortical ischemic areas (1) respectively 5min and 6h after cerebral ischemia, electroacupuncture intervention in MCAO rats, "Baihui", "Shen Ting" point. (2) through the memory of the condition of fear and the score of nerve function defect and cerebral infarction volume, the evaluation of the "hundred meetings" of electroacupuncture, "God court" to cerebral ischemia rats Neuroprotective effect. (3) Nissl staining and fluorescence staining were used to observe the injury of cerebral ischemia tissue, Cofilin rod formation, apoptosis and neuron loss. (4) the activity of Caspase-3 in the ischemic penumbra and the expression level of Cofilin and p-cofilin in the cytoplasm and mitochondria were detected by Western blot. Results: the first part (1) ATP consumption The depletion can induce the formation of Cofilin rod and apoptosis in the primary hippocampal neurons. (2) TTC and NeuN staining showed that the infarct volume increased from 2H to 24h, and then 24h to 7d after MCAO. (3) Rod structure was generated in the 2H and semi dark band of the infarct core area, then reached its peak, and then decreased. Induced MAP2 degradation and apoptosis in infarct cortex after cerebral apoplexy. (4) overexpression of Limk1 inhibits the activation of Cofilin, which can significantly reduce the formation of Rod in the dark zone and MAP2 degradation in the ischemic zone of MCAO rats, and reduce the apoptosis of Cofilin mediated cells, and further confirm the role of Cofilin in cerebral ischemia. The second part (1) the acute phase of electroacupuncture "hundred meetings", "the divine court" "Acupoint treatment of MCAO rats can significantly reduce the volume of cerebral infarction and improve the neurological deficits and cognitive impairment caused by brain damage. (2) Western blot analysis shows that the two points of electroacupuncture can also significantly inhibit the mitochondrial translocation and Caspase-3 cutting of Cofilin. (3) the results of double immunofluorescent labeling suggest that the Electroacupuncture of" 100 meetings "," "Acupoint therapy" not only partially inhibits Cofilin rod formation and MAP2 degradation, but also significantly reduces cerebral tissue damage induced by ischemic stroke, cell apoptosis and neuron loss. Conclusion: (1) in vivo and in vitro ischemia can induce the formation of Cofilin rod and neuronal cell death, while Limk1 overexpression inhibits Cofilin overactivation, which is significant Reduce the formation of Rod formation, MAP2 degradation and Cofilin mitochondrial Translocation Induced Apoptosis after cerebral ischemia. Thus, Cofilin plays an important role in Rod formation and cell apoptosis after ischemic stroke. It can be used as a potential therapeutic target for ischemic stroke. (2) the acute phase electroacupuncture "hundred meetings", "Shen Ting" point can reduce the formation of Cofilin rod and MA P2 degradation, inhibiting the mitochondrial translocation of Cofilin, alleviates brain damage and apoptosis of ischemic stroke, thus promoting the recovery of cognitive function, which may be a new mechanism of electroacupuncture therapy.

【学位授予单位】：福建中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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