异丙酚对大鼠认知功能及不同脑区磷酸化蛋白表达的影响

发布时间：2018-04-20 19:58

本文选题：异丙酚 + 空间学习记忆　；参考：《南方医科大学》2014年硕士论文

【摘要】：背景麻醉药物和镇静药物对于神经系统的损害作用一直以来都是麻醉领域的研究热点,异丙酚(propofol)是一种新型的短效静脉麻醉药物,广泛应用于临床麻醉诱导、维持及ICU镇静,具有起效快,麻醉状态可控性强,维持时间短,苏醒迅速,无积蓄,副作用小等优点。已有学者发现异丙酚可阻断人的工作记忆,并可造成健忘症的发生,但是Lee等的研究却发现单次输注异丙酚并不会引起成年和老年大鼠的记忆损害。然而在临床上异丙酚的用途不仅是单次输注,在全凭静脉麻醉时及ICU内可作为连续或间断使用达数次或数小时乃至数天之久,因此仅凭单次输注来判断异丙酚是否具有学习、记忆能力损害作用并无实际意义。目前的临床证据尚不足以证明异丙酚与学习、记忆能力损害之间存在因果关系,而临床用药的多样性必然使得观察人的神经损害与异丙酚的关系复杂化,因此在积累临床数据的同时,有必要通过进一步的动物实验来研究异丙酚和神经损害之间的关系和相应的机制。哺乳动物成年神经再生的发现颠覆了长久以来认为成年大脑不能产生新生神经元的观点。成年神经再生,起源于神经前体细胞,可在成年大脑的两个区域发生：侧脑室的脑室下区和海马齿状回区的背侧区。成年神经再生是神经干细胞的增殖、分化、成熟及向神经回路整合,产生新生神经元的过程。海马是哺乳动物边缘系统的一个重要的结构,海马的结构内部可分为齿状回、CA1区、CA3区三个主要组成部分。海马齿状回区是哺乳动物成年期新生神经元生成的几个脑区之一,是在记忆过程尤其是建立和应用空间学习记忆时发挥重要作用的神经集成网络的重要组分。迄今为止,已知的麻醉药物的作用机制主要是调控中枢神经系统关键部位的突触传递和神经细胞膜上的离子通道。异丙酚也主要作用于突触,调节突触前膜递质的释放和突触前后膜Y-氨基丁酸受体的功能,从而产生中枢抑制效应,发挥麻醉作用。但是异丙酚具体作用的分子机制还不清楚。大部分在突触传递中发挥重要作用的神经递质和离子通道都是蛋白质。蛋白质是生命活动的最主要和最直接的体现者和执行者,也是多种致病因素和药物作用的靶分子。蛋白质修饰尤其是磷酸化和去磷酸化作用,在不同的细胞功能如细胞分化、细胞生长、细胞凋亡等发挥重要作用。研究表明,阿尔茨海默病的脑组织的tau蛋白异常磷酸化作用发生在神经纤维缠结之前,而且,经过异氟醚和地氟醚吸入的动物体内tau蛋白磷酸化作用增强,这种作用可能导致麻醉后短期认知功能障碍。因此发现异丙酚输注后不同脑区磷酸化蛋白的水平的而变化对探讨异丙酚的可能的神经毒性机制是非常重要的。大脑是一种高度交互的实体,一些独立的大脑区域协作发挥生物学功能。丘脑被认为是皮质下区和大脑皮层的信息传递部位。而且,功能性脑成像也证实丘脑是麻醉药作用的主要脑区。研究显示海马主要负责心理思维过程,比如最初的学习记忆能力也包括意识行为。Wei等曾报道异丙酚可能由于其对学习记忆能力的影响,从而促进大鼠海马CA1区突触的长时程抑制的发展。大脑皮层是唤醒系统的最后的靶向脑区,同时背外侧的前额皮质是大脑皮层最重要的部分,参与许多生理过程,如情感认知,随意运动,工作记忆,甚至维持哺乳动物的激活状态。本研究第一部分通过Morris水迷宫实验和免疫荧光技术及激光共聚焦技术来观察异丙酚重复镇静对大鼠空间学习记忆能力和海马齿状回新生神经元的影响,进一步探讨异丙酚可能存在的神经毒性作用。第二部分通过从异丙酚麻醉的动物模型中分离提取丘脑、海马、额叶的磷酸化蛋白,然后用二维电泳和质谱分析法筛选及鉴定异丙酚作用于不同脑区后对磷酸化蛋白表达水平的影响,为后续研究其相应的机制奠定基础。材料与方法 1.SD成年雄性大鼠分别腹腔给予异丙酚或脂肪乳,每天2次,持续7天。首先,应用Morries水迷宫实验观察和检测大鼠给药后28天的空间学习和记忆能力。其次,给药后,经腹腔注射BrdU标记,分别计数给药后1天、14天和28天大鼠海马齿状回颗粒下区BrdU阳性细胞数。最后,应用激光共聚焦显微镜扫描给药后14天大鼠新生神经元的树突长度和分枝数目。 2.SD成年雄性大鼠分别尾静脉给予异丙酚或脂肪乳,20min后断头取丘脑、海马、额叶。首先提取三个脑区磷酸化蛋白后双向凝胶电泳检测差异表达的磷酸化蛋白,然后用MALDI-TOF MS鉴定并在数据库中搜索。最后Western Blot验证差异表达的磷酸化蛋白。结果 1.异丙酚镇静28天后,大鼠的逃逸潜伏期、游泳距离明显延长,第5天在目标区域的停留时间明显减少。异丙酚在给药后第1天BrdU阳性细胞数无显著差异,但是第14天和第28天则显著减少。在给药后第14天大鼠海马齿状回颗粒下区异丙酚组的DCX阳性细胞数显著少于溶媒对照组,异丙酚组的DCX阳性新生神经元的树突长度和分枝数目显著低于溶媒对照组。 2.双向凝胶电泳显示丘脑、海马、额叶这三个脑区共有21个蛋白位点显著不同,其中经MALDI-TOF MS成功鉴定的有16个磷酸化蛋白位点。讨论海马是中枢神经系统中参与认知功能如学习记忆能力等功能的最主要部位,海马齿状回区是生命活动中不断产生新生神经元的一个重要脑区。Deng等人综述了许多关于新生神经元对学习和记忆能力影响的报道,然而这些报道有些认为减少神经再生会损害海马依赖的学习和记忆能力,另一派则认为神经再生的减少并不发挥什么作用。以往也有报道认为空间学习能促进新生神经元增殖,增加新生神经元的存活率,亦可能减少新生神经元的数量。因此,本研究旨在进一步探讨新生神经元与空间学习记忆能力的关系,以及异丙酚作用对其的影响。这些新生神经元经过神经前体细胞的不断增殖,形成神经连接和突触,逐渐成为具有记忆能力的成熟的神经元。它们主要位于海马齿状回区,大约4周后具有空间学习的能力。我们的结果发现异丙酚作用的大鼠在给药后4周开始出现行为学损害,这也恰好是新生颗粒细胞具备参与到现有神经环路中的能力的时候。并且,我们在给药后分别计数了2组大鼠颗粒下区的BrdU阳性细胞数。在给药后1天,两组BrdU阳性细胞数的差异无统计学意义,说明异丙酚对于海马成年神经干细胞的增殖并无影响。然而在给药后28天,异丙酚重复镇静组的大鼠颗粒下区BrdU阳性细胞数则显著低于溶媒对照组,提示异丙酚可能损伤的是增殖中的细胞。这些结果表明异丙酚重复镇静可损害成年神经再生,尤其是与学习能力密切相关的神经前体细胞的存活,进而影响由神经前体细胞增殖分化而成的神经元的数量。微管相关蛋白(doublecortin,DCX)作为神经元前体细胞的标志物可以用来研究神经元前体细胞的增殖和迁移。我们选择DCX——成年海马齿状回新生神经元可靠的标志物,对其进行免疫染色。并通过激光共聚焦显微镜比较了两组DCX阳性新生神经元的数量和发育状态。异丙酚镇静组的DCX阳性细胞数在给药后14天显著减少,与BrdU阳性细胞计数的结果一致,表明该组神经前体细胞存活较少。同时,该组的新生神经元在形态学上的成熟度显著延迟。我们的结果表明异丙酚镇静组新生神经元的树突结构的复杂度,无论是在长度、分枝数目上均显著减少。在神经元的发育过程中,树突结构的复杂度是其成熟度的一个重要标志,同时也能够说明其未来的学习能力。因此,由异丙酚镇静所造成的成年神经再生能力的下降,不仅表现在神经元的数量上,而且也造成了神经元树突成熟度的下降,由此亦不难推断出其导致的行为学损害。我们观察了异丙酚重复镇静可损害大鼠空间学习和海马区成年神经再生,但异丙酚的这一作用并非通过损害成年神经干细胞增殖,而是通过影响其存活率以及新生神经元的树突结构引起的。但是异丙酚应用后所表现出的这种神经毒性的具体机制还有待于进一步研究。在本研究中我们采用蛋白质组学方法筛选异丙酚作用后对丘脑、海马、额叶中磷酸化蛋白的差异表达,经过生物信息学分析和Western Blot验证去探讨异丙酚可能的作用机制。实验结果发现异丙酚作用后有16种磷酸化蛋白表达水平存在差异,对差异表达的磷酸化蛋白进行生物信息学分析后,共发现6种蛋白质：细胞角蛋白18(KRT18)、凝溶胶蛋白(GSN)、微管蛋白(TbB2C)、巨噬细胞加帽蛋白(MCP)、肌动蛋白(Actin)及载脂蛋白E(ApoE)可能与异丙酚的神经毒性作用相关,这些蛋白质直接或间接的参与构成了细胞骨架及其稳定性的维持。 Tau蛋白是中枢神经系统神经元内富含的II型微管结合蛋白(MAPs),它的磷酸化状态可被一群特定的磷酸酶和磷酸激酶调节,这些酶对维持神经元的功能和发育有重要作用。Tau蛋白的高度磷酸化被认为与阿尔茨海默病(AD)和术后认知功能障碍的发病机制相联系。七氟醚、异氟醚和异丙酚均可导致tau蛋白高度磷酸化,这可能可以解释术后认知功能障碍的发生。ApoE也可在AD患者特有的淀粉样斑块和神经纤维缠结中检测到,参与AD的发病过程。ApoE4转基因小鼠脑内过度磷酸化的Tau蛋白的单体和多体的沉积表明ApoE能够影响tau磷酸化的水平。我们推测异丙酚麻醉引起的术后认知功能障碍可能是由于血清ApoE水平变化导致的。然而这些因素的相互关系和ApoE磷酸化是否参与这个过程是未来需要探索的。生物信息学分析显示GSN和KRT18对酒精有反应。酒精能够削弱机体对外界刺激的反应,类似于异丙酚麻醉后出现的行为学改变。氨基酸神经递质受体在酒精依赖中起重要作用。酒精是GABA受体激动剂和NMDA受体拮抗剂,在大脑发育期通过抑制ERK磷酸化从而导致神经系统的退行性变。大脑发育期酒精对大脑干细胞凋亡的影响作用也类似于异丙酚。海马是大脑对酒精损害最敏感的区域,也是异丙酚麻醉作用的靶区域,谷氨酸是海马环路输入和输出的主要神经递质。总之,异丙酚的靶向区域和对信号传递的影响都与酒精相似。研究还表明酒精可能引起肝肾KRT18的磷酸化。我们的研究发现,相比对照组,异丙酚给药组的大鼠海马区KRT18也发生了磷酸化改变。但是,具体的作用机制还需要进一步研究。总而言之,本研究采用蛋白质组学方法发现了丘脑、海马、额叶的16种差异表达的磷酸化蛋白。同时使用生物信息学分析差异表达的磷酸化蛋白的共同特征去探讨异丙酚作用于神经系统的可能机制为进一步阐明异丙酚的神经毒性机制提供了很有意义的参考。结论 1.Morris水迷宫实验表明异丙酚重复镇静可损害大鼠空间学习记忆能力,还可造成海马齿状回区的新生神经元的数量下降以及树突发育的不成熟,进而损害学习记忆能力。 2.异丙酚麻醉作用后可引起丘脑、海马、额叶的KRT18,GSN, TbB2C, MCP,肌动蛋白,ApoE等细胞骨架蛋白质的差异表达,这种异丙酚引起的蛋白质磷酸化为未来研究异丙酚的神经毒性机制提供了支持。
[Abstract]:background
The effect of narcotic drugs and sedative drugs on the nervous system has always been a hot spot in the field of anesthesia. Propofol (propofol) is a new type of short effective intravenous anesthetic. It is widely used in clinical anesthesia induction, maintenance and ICU sedation with rapid onset, strong controllability of narcotic state, short maintenance time, rapid recovery and no accumulation. It has the advantages of small side effect and so on.
Some researchers have found that propofol can block working memory and cause amnesia, but Lee studies have found that a single infusion of propofol does not cause memory damage in adult and old rats. However, the use of propofol in clinical use is not only a single infusion, but also in the case of intravenous anesthesia and in ICU. There is no practical significance in judging whether propofol is learning, and memory impairment is not practical. Current clinical evidence is not yet sufficient to demonstrate the relationship between propofol and learning and memory impairment, and the diversity of clinical medication inevitably leads to the view. The relationship between nerve damage and propofol is complex, so it is necessary to study the relationship and mechanism between propofol and nerve damage by further animal experiments while accumulating clinical data.
The discovery of adult nerve regeneration in mammals overturns the long-standing view that the adult brain does not produce new neurons. The adult nerve regeneration, originated from the neural precursor cells, can occur in two regions of the adult brain: the dorsal ventricle of the ventricle and the dorsal region of the dentate gyrus of the hippocampus. Adult neural regeneration is a neural stem cell. The hippocampus is an important structure of the mammalian marginal system. The internal structure of the hippocampus can be divided into three main parts of the dentate gyrus, CA1 area and CA3 region. The dentate gyrus of the hippocampus is one of the several brain regions of the newborn neurons of mammalian adults. It is an important component of the neural network, which plays an important role in the memory process, especially in the establishment and application of spatial learning and memory. So far, the mechanism of the known narcotic drugs is mainly to regulate the synaptic transmission in key parts of the central nervous system and the ion channels on the membrane of the nerve cell. The release of the transmitters of the presynaptic membrane and the function of the Y- aminobutyric acid receptor in the postsynaptic membrane produce the central inhibitory effect and play an anesthetic effect. However, the molecular mechanism of the specific action of propofol is not clear. Most of the neurotransmitters and ion channels, which play an important role in synaptic transmission, are proteins. The most important and direct embodying and executor of movement are also the target molecules of a variety of pathogenic factors and drug effects. Protein modification, especially phosphorylation and dephosphorylation, plays an important role in different cellular functions such as cell differentiation, cell growth, cell apoptosis and so on. Studies have shown that the tau eggs of Alzheimer's disease are in the brain tissue. White abnormal phosphorylation occurs before neurofibrillary tangles, and the enhanced phosphorylation of tau protein in animals inhaled by isoflurane and desflurane may lead to short-term cognitive impairment after anesthesia. Therefore, the changes in the level of phosphorylated protein in different brain regions after propofol infusion are found to be discussed in the study of propofol. The possible neurotoxicity mechanism is very important.
The brain is a highly interactive entity, and some independent brain regions collaborate to perform biological functions. The thalamus is considered to be the information transfer site of the subcortical and cerebral cortex. Moreover, functional brain imaging also confirms that the thalamus is the main brain area for the effect of anesthetics. Learning and memory ability also includes the awareness behavior.Wei and other reports that propofol may be affected by its learning and memory ability, thus promoting the development of long term depression in the synapse in the hippocampus CA1 region. The cerebral cortex is the final target brain area of the awakening system, while the dorsal frontal cortex is the most important part of the cerebral cortex, and participates in the involvement of the cerebral cortex. Many physiological processes, such as emotional cognition, voluntary movement, working memory, and even maintain the activation state of mammals.
In the first part of this study, the Morris water maze test, immunofluorescence technique and laser confocal technique were used to observe the effect of propofol repeated sedation on the spatial learning and memory ability of rats and the hippocampal gyrus rebirth neurons, and further explore the possible neurotoxicity of propofol. The second part passed the action of propofol anesthesia. The phosphorylated protein of the thalamus, hippocampus and frontal lobe was isolated and extracted from the model. Then two-dimensional electrophoresis and mass spectrometry analysis were used to screen and identify the effect of propofol on the expression of phosphorylated protein after the action of propofol on different brain regions, which laid the foundation for the subsequent study of its corresponding mechanism.
Materials and methods
1.SD adult male rats were given intraperitoneal injection of propofol or fat milk 2 times a day for 7 days. First, the Morries water maze test was used to observe and detect the spatial learning and memory ability of the rats at 28 days after administration. Second, after the administration, the BrdU markers were injected into the abdominal cavity for 1 days, 14 days and 28 days after the administration of the rat sea dentate gyrus. The number of BrdU positive cells in the region was measured. Finally, the number of dendritic cells and the number of branches of the newborn neurons were examined by confocal laser scanning microscope on the 14 day after administration.
The adult male rats of 2.SD were given propofol or fat milk respectively. After 20min, the thalamus, hippocampus and frontal lobe were taken out of the head. First, three phosphorylated proteins in the brain regions were extracted by two-dimensional gel electrophoresis to detect the differential expression of phosphorylated protein. Then, MALDI-TOF MS was identified and searched in the database. Finally, Western Blot was used to verify the differential expression of phosphoric acid. Protein.
Result
1. after 28 days of propofol sedation, the escape latency, swimming distance of the rats were obviously prolonged, and the stay time in the target area decreased significantly on the fifth day in the target area. The number of BrdU positive cells in the first days after the administration of propofol was not significantly different, but the fourteenth and twenty-eighth days decreased significantly. At fourteenth days after the administration, the D of the propofol group in the dentate gyrus of the rats The number of CX positive cells was significantly less than that of the control group. The number of dendritic cells and branches of DCX positive neurons in the propofol group was significantly lower than that in the solvent control group.
2. bi-directional gel electrophoresis showed that there were 21 different protein loci in the three brain regions of the thalamus, hippocampus and frontal lobe, of which 16 phosphorylated protein sites were identified by MALDI-TOF MS.
discuss
Hippocampus is the most important part of cognitive function, such as learning and memory ability in the central nervous system. The hippocampal dentate gyrus is an important brain area,.Deng, which produces new neurons in life activities. Many reports about the effects of new neurons on learning and memory ability are reviewed. However, these reports suggest that some of these reports have been reported. The reduction of nerve regeneration can damage the learning and memory ability of the hippocampus, while the other believes that the reduction of nerve regeneration does not play a role.
Therefore, the purpose of this study is to further explore the relationship between new neurons and spatial learning and memory, and the effect of propofol on them. These new neurons, through the continuous proliferation of neural precursor cells, form neural connections and synapses, and gradually become mature neurons with memory ability. They are mainly located in the dentate dentate shape of the hippocampus. Back area, about 4 weeks later, has the ability to learn in space. Our results found that the propofol action rats began to appear behavioral damage at 4 weeks after the administration, which was also the time when new granulosa cells had the ability to participate in the existing neural circuits. And we counted the BrdU Yang of the subregion of the 2 groups of rats after the drug was given. The number of BrdU positive cells in the two groups was not statistically significant at 1 days after the administration, indicating that propofol had no effect on the proliferation of adult neural stem cells in the hippocampus. However, the number of BrdU positive cells in the subgranular subgranular area of the propofol repeated sedative group was significantly lower than that of the dissolvent control group at 28 days after the administration, suggesting that propofol might be damaged. These results suggest that the repeated sedation of propofol can damage the regeneration of the adult nerve, especially the survival of the neural precursor cells, which are closely related to the learning ability, and then affect the number of neurons formed by the proliferation and differentiation of the neural precursor cells.
Doublecortin (DCX), as a marker of neuronal precursor cells, can be used to study the proliferation and migration of neuronal precursor cells. We choose DCX, a reliable marker of adult hippocampal dentate gyrus, and immunize them. And two groups of DCX positive freshmen are compared by light confocal microscopy. The number and development state of the neurons. The number of DCX positive cells in the propofol sedative group decreased significantly at 14 days after the administration, consistent with the results of the BrdU positive cell count, indicating that the group of neural precursor cells survived less. The complexity of the dendritic structure of newborn neurons, both in length and number of branches, is significantly reduced. In the development of neurons, the complexity of the dendritic structure is an important sign of its maturity, and it can also indicate its future learning ability. Therefore, the adult nerve regeneration ability caused by isopropanol sedation The decline is not only manifested in the number of neurons, but also causes the decline of dendrite maturity, so it is not difficult to deduce the behavioral damage caused by it.
We observed that propofol repeated sedation could damage spatial learning and adult neural regeneration in the hippocampus, but this effect of propofol is not by damaging the proliferation of adult neural stem cells, but by affecting the survival rate and the structure of the dendrites of newborn neurons. However, the neurotoxicity of propofol is shown after the use of propofol. The specific mechanism of sex remains to be further studied.
In this study, we used proteomic methods to screen the differential expression of phosphorylated proteins in the thalamus, hippocampus and frontal lobe after propofol action. Through bioinformatics analysis and Western Blot verification, we explored the possible mechanism of propofol. The experimental results showed that the expression level of 16 kinds of phosphorylated proteins was poor after the use of propofol. After bioinformatics analysis of differentially expressed phosphorylated proteins, 6 proteins were found: cytokeratin 18 (KRT18), GSN, microtubule protein (TbB2C), macrophage plus CAP protein (MCP), actin (Actin) and apolipoprotein E (ApoE), which may be related to the neurotoxicity of propofol. These proteins are direct Or indirect participation constitutes the maintenance of cytoskeleton and its stability.
Tau protein is a type of II microtubule binding protein (MAPs) rich in neurons in the central nervous system. Its phosphorylation state can be regulated by a group of specific phosphatase and phosphokinase. These enzymes play an important role in maintaining the function and development of neurons. The high phosphorylation of.Tau protein is considered to be associated with Alzheimer's disease (AD) and postoperative cognitive function. The pathogenesis of the disorder is linked. Sevoflurane, isoflurane, and propofol can lead to high phosphorylation of tau protein, which may explain the occurrence of postoperative cognitive dysfunction,.ApoE can also be detected in the amyloid plaques and neurofibrillary tangles endemic to AD patients, and is involved in the excessive phosphorylation of the brain in the.ApoE4 transgenic mice with the pathogenesis of AD. The deposition of the monomers and multibodies of Tau protein indicates that ApoE can affect the level of tau phosphorylation. We speculate that the postoperative cognitive dysfunction caused by propofol anesthesia may be caused by changes in serum ApoE levels. However, the relationship between these factors and whether the ApoE phosphorylation is involved in this process is to be explored in the future.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

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