右美托咪定对大鼠海马神经元缺氧复氧损伤的作用及其机制

发布时间：2018-05-12 04:34

本文选题：右美托咪定 + 钙超载　；参考：《青岛大学》2017年博士论文

【摘要】：脑缺血是脑部组织血流量不足的一种疾病,这种情况通常可以导致脑组织的代谢率以及能量的降低,和随之引起来的局部区域的脑梗死。当脑缺血这种情况发生时,由于来自于血液的氧以及营养物的供给减少,可能使脑部的神经元组织产生饥饿感。饥饿可以诱导细胞产生凋亡,它有两个转移方向,一是往好的方向转化:即恢复生存,二是往坏的方向转化:即进入死亡。从病理生理学上来讲,再灌注损伤通常发生在缺血期后血液供应重新返回脑组织时。与缺血性损伤相比,局部缺血/再灌注(I/R)可能是引起线粒体呼吸链反应的破坏以及脑部组织脑损伤区域中炎性介质的过量产生以及包浆中的Ca2+离子大量沉积从而对神经元造成的更多的损害;在临床外科上,有包括急性缺血性中风以及急性创伤性脑损伤等很多种脑部疾病,都可以引起缺血以及再灌注损伤。再灌注损伤在临床上被认为是继发性脑损伤的重要因素。线粒体在调节细胞生命活动过程中起着重要的作用,它在细胞中以较高的速度不停的运动着,通过自我分裂或者彼此间相互融合,实现新个体的产生,并可以在细胞中形成一种紧密的联系。线粒体通常被人们称作“细胞动力工厂”,其分裂和融合是否平衡,影响着功能和结构的变化。经过以前的研究表明,线粒体可以减少细胞色素C、以及调亡蛋白AFI因子等的表达,从而在细胞的程序性死亡中起着重要的调控作用。我们通过对大鼠短暂性脑I/R损伤模型的分析表明,线粒体分裂的多少与细胞凋亡的程度有着密切的关联,抑制线粒体的分裂能够减轻脑皮质部分的I/R损伤。另外,有研究证实:线粒体的分裂过程中,钙信号通路可以对线粒体分裂相关蛋白Drpl的磷酸化与去磷酸化产生影响,进而对线粒体的分裂起到调控作用;因此如果减少线粒体摄取钙离子的量,就可以降低线粒体的分裂能力,减少分裂线粒体的分裂次数;在我们以前的实验中也曾发现,降低MCU活性,对脑梗死面积及自由氧产生有一定的抑制作用,减轻线粒体肿胀损伤。这点在大鼠短暂性I/R损伤模型中得到了很好的体现。右美托咪定是一种通过选择性激动发挥其作用的镇静剂以及镇痛剂,主要作用于α2肾上腺素受体。以前的研究已经证明过,右美托咪定可防止缺血再灌注损伤诱导的对心肌,肾,肠,肝,肺以及脑的损伤。同时Kuhmonen等人发现右美托咪定能够对大鼠中脑动脉闭塞引起的脑梗死有很大的益处。右美托咪定还可以减少炎性介质诱导的Ca2+释放以及钙超载减少炎症的继续进展。最新研究表明右美托咪定可以抑制氧糖剥夺或者Ca2+诱导线粒体肿胀而保持线粒体形态与功能的稳定,抑制海马神经元钙超载,减少钙调神经磷酸酶的激活,进而抑制线粒体分裂,其机制可能是通过抑制钙调神经磷酸酶的活性,减少其对Drp1-ser637的磷酸化,进而抑制Drp1的转位,减少与线粒体外膜蛋白Fis1的结合;另外,它还可以抑制Drp1-ser637与Fis1的表达,减少它们在线粒体外膜的共定位结合,从而抑制线粒体分裂的启动。凋亡是I/R诱导的神经元损伤中的主要途径。线粒体在涉及细胞凋亡调节的内在途径中起重要作用。I/R诱发引起来炎症因子可以引起线粒体的结构和功能异常,导致Cyt C从线粒体释放到细胞质中和随后级联激活半胱天冬酶-9,-3和-6,进而引起相关的凋亡级联反应,Caspase-3的激活是线粒体凋亡必须经历的过程,它通过与其他家族成员的级联反应,可以加速细胞的凋亡。因此我们就假设右美托咪定可以通过抑制钙超载来减少线粒体分裂、保持线粒体形态与功能的稳定,进而通过抑制线粒体-细胞色素C-半胱氨酸蛋白酶凋亡途径,来减少神经元细胞的凋亡,从而发挥脑保护作用。目的:探讨右美托咪定在大鼠海马神经元缺氧复氧损伤中的作用,以及右美托咪定对线粒体分裂的的影响。方法:新生24h之内的SD大鼠,断头分离大脑海马区神经组织,收集获得神经元细胞进行原代海马神经元培养,培养至第8天,氧糖剥夺法建立海马神经元缺氧复氧模型,按照随机数字表法将其随机分为6组::空白对照组(C组),细胞未给予任何处理;赋形剂组(V组),赋形剂二甲基亚砜(DMSO),终浓度为0.01%]加入细胞培养基;缺氧复氧组(H/R组);右美托咪定组:D1、D2、D3组,在细胞缺氧复氧期间分别加入右美托咪定0.1、1、10μmol/L。分组处理之后,用细胞增殖与毒性检测试剂盒检测各组细胞活性(确定右美托咪定合适的浓度范围)、用透射电镜观察线粒体的超微结构、用激光共聚焦显微镜观察各组神经元细胞质Ca2+荧光强度、用ELISA法检测细胞钙调神经磷酸酶活性、用Western-blot检测Drp1、Fis1、Cyt C、capase3蛋白的表达。用流式细胞学检测神经元细胞的凋亡率。结果:与对照组比较,H/R组的细胞数目以及活性显著减少,与H/R组比较,右美托咪定组(D1、D2、D3)细胞数量相对增加,并且D2组的细胞活性比D1和D3相对要高。与对照组相比,H/R组的细胞凋亡率显著增多,与H/R组比较,右美托咪定组(D1、D2、D3)细胞凋亡率相对降低,并且D2组的细胞凋亡率比D1和D3相对要低。与对照组相比,神经元Ca2+荧光强度、钙调神经磷酸酶活性及Drp1、Fis1、Cyt C、caspase3蛋白的表达升高(P0.05);与H/R组比较,D1、D2、D3组神经元活性增强、线粒体超微结构破坏明减轻,神经元Ca2+荧光强度、钙调神经磷酸酶活性及Drp1、Fis1、Cyt C、caspase3的表达均降低(P0.05);D2组又较D1、D3组细胞活性增强,细胞质Ca2+荧光强度、钙调神经磷酸酶活性及Drp1、Fis1、Cyt C、caspase3的表达均降低(P0.05)。结论:右美托咪定0.1、1、10μmol/L可以明显改善大鼠海马神经元在缺氧复氧中的损伤,其中1μmol/L是最佳的保护浓度,其机制可能是与右美托咪定抑制钙超载介导的线粒体分裂及线粒体凋亡途径有关系。
[Abstract]:Cerebral ischemia is a disease of the brain tissue with insufficient blood flow, which usually leads to the metabolic rate of brain tissue and the decrease of energy, and the consequent cerebral infarction in the local area. When the condition of cerebral ischemia occurs, the brain tissue may be caused by the reduction of oxygen from the blood and the supply of nutrients. Starvation produces a sense of hunger. Starvation can induce cell apoptosis. It has two direction of metastasis, one is to turn in a good direction, that is to restore survival, and the two is to turn into the bad direction: into death. From pathophysiology, reperfusion injury usually occurs after the ischemia period, and the blood supply returns to the brain tissue. Local ischemia / reperfusion (I/R) may be the damage to the mitochondrial respiratory chain reaction and the excessive production of inflammatory mediators in the brain tissue of the brain tissue and the deposition of Ca2+ ions in the plasma in the plasma, thus causing more damage to the neurons. In clinical surgery, acute ischemic stroke and acute traumatic brain damage are included. Many kinds of brain diseases, such as injuries, can cause ischemia and reperfusion injury. Reperfusion injury is considered to be an important factor in secondary brain damage in clinical. Mitochondria play an important role in regulating cell life activities. It is moving at a high speed in cells, by self splitting or by each other. Interfusion, the production of new individuals, and the formation of a close association in cells. Mitochondria are often called "cell power plants", and their division and fusion are balanced, affecting the changes in function and structure. After previous studies, mitochondria can reduce cytochrome C, and AFI factor, and so on. Expression, which plays an important role in the programmed cell death of the cell, has been analyzed by the analysis of the transient brain I/R damage model in rats. It is shown that the number of mitochondrial divisions is closely related to the degree of apoptosis, and the inhibition of mitochondrial division can reduce the I/R damage in the cortical part of the brain. The calcium signaling pathway can affect the phosphorylation and dephosphorylation of mitochondrial mitotic protein Drpl and regulate the mitosis of mitochondria. Therefore, if the amount of calcium ions in mitochondria is reduced, the splitting ability of mitochondria can be reduced and the number of split mitochondria is reduced. In our previous experiments, it was also found that reducing the activity of MCU has a certain inhibitory effect on the area of cerebral infarction and the production of free oxygen and alleviated the swelling and injury of mitochondria. This is a good manifestation of the transient I/R damage model in rats. Right metoimidin is a sedative and analgesic agent that plays its role by selective excitation. A previous study has shown that dexmedetomidine can prevent damage to the myocardium, kidney, intestines, liver, lungs, and brain induced by ischemia reperfusion injury. Kuhmonen et al. Found that right metoimidin could be of great benefit to cerebral infarction caused by occlusion of the middle cerebral artery in rats. Right metomomidin can also be used. Reduction of inflammatory mediators induced Ca2+ release and calcium overload to reduce inflammation. The latest research shows that dexmedetomidine can inhibit oxygen glucose deprivation or Ca2+ induced mitochondrial swelling to maintain mitochondrial morphologic and functional stability, inhibit calcium overload in hippocampal neurons, reduce the activation of calcineurin, and then inhibit line particles. The mechanism may be to inhibit the phosphorylation of Drp1-ser637 by inhibiting the activity of calcineurin, thus inhibiting the transposition of Drp1 and reducing the binding of the mitochondrial membrane protein Fis1. In addition, it also inhibits the expression of Drp1-ser637 and Fis1, and reduces the co localization of the mitochondria in the outer membrane of the mitochondria, and thus inhibits the line particles. Apoptosis is the main pathway of I/R induced neuronal damage. Mitochondria play an important role in the internal pathway involving apoptosis regulation..I/R induces the structure and function of mitochondria caused by inflammatory factors, resulting in the release of Cyt C from mitochondria to cytoplasm and subsequent cascade activation of cysteine and asparagus. The enzyme -9, -3 and -6 cause the associated apoptosis cascade reaction, and the activation of Caspase-3 is the necessary process of mitochondrial apoptosis. It can accelerate cell apoptosis by cascade reaction with other family members. Therefore, we hypothesized that dexmedetomidin could reduce mitochondrial division by inhibiting calcium overload and maintain mitochondrial morphology. Function stability, and then by inhibiting the mitochondrial cytochrome C- cysteine protease apoptosis pathway, to reduce the apoptosis of neuron cells and to play the role of brain protection. Objective: To explore the role of dexmedetomidine in the hypoxia reoxygenation injury of rat hippocampal neurons, and the effect of right metoimidin on mitochondrial division. In the SD rats of 24h, the nerve tissue in the hippocampus of the brain was separated and the neuron cells were collected and cultured for eighth days. The oxygen deprivation method was used to establish the hypoxic reoxygenation model of hippocampal neurons. According to the random number table method, the neurons were randomly divided into 6 groups: the blank control group (group C), the cells were not given any treatment; the cells were not treated with any treatment; The form agent group (group V), excipient two methyl sulfoxide (DMSO), the final concentration was 0.01%] added to cell culture medium, anoxic reoxygenation group (group H/R), right metoimidin group: D1, D2, D3 group, after cell hypoxia reoxygenation was added to right metomomidin 0.1,1,10 u mol/L. group treatment, the cell activity was detected by cell proliferation and toxicity detection kit. The ultrastructure of mitochondria was observed by transmission electron microscopy. The cytoplasmic Ca2+ fluorescence intensity of each group was observed by laser confocal microscopy. The activity of calcineurin was detected by ELISA. The expression of Drp1, Fis1, Cyt C, capase3 protein was detected by Western-blot. Flow cytometry was used to detect the expression of the protein. Compared with the control group, the number and activity of the cells in the H/R group decreased significantly. Compared with the H/R group, the number of D1, D2, D3 cells in the right metoimidin group increased relatively, and the cell activity of the D2 group was higher than that of the D1 and D3. Compared with the control group, the apoptosis rate of the H /R group was significantly increased, compared with the H/R group, right metoami was compared. The apoptosis rate of D1, D2, D3 was relatively lower, and the apoptosis rate of D2 group was lower than that of D1 and D3. Compared with the control group, the neuronal Ca2+ fluorescence intensity, calcineurin activity and Drp1, Fis1, Cyt C, and the expression of Caspase3 protein increased. The fluorescence intensity of Ca2+, the activity of calcineurin and the expression of Drp1, Fis1, Cyt C, Caspase3 were all decreased (P0.05), and the activity of the D2 group was more than D1, and the cell activity of the D3 group was enhanced, the cytoplasm Ca2+ fluorescence intensity, the activity of calcineurin and the Drp1, and the expression were all decreased. L can significantly improve the damage of rat hippocampal neurons in hypoxia and reoxygenation, in which 1 mol/L is the best protective concentration. The mechanism may be related to the pathway of mitochondria division and mitochondrial apoptosis mediated by dexmedetomidine inhibiting calcium overload.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R614

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