α-硫辛酸对创伤性癫痫大鼠的脑保护作用及其机制研究

发布时间：2018-06-15 15:02

本文选题：颅脑创伤 + 创伤性癫痫　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:癫痫是一种慢性神经系统疾病,具有一过性、刻板性、突发突止的特点。创伤性脑损伤(traumatic brain injury,TBI)是症状性癫痫的重要病因之一。癫痫发作引起的脑缺血缺氧反应使大脑神经元受损,产生大量自由基,造成线粒体损伤,而线粒体损伤引起的细胞凋亡和坏死,使得癫痫发作进一步加重。本研究通过建立创伤性癫痫(posttraumatic epilepsy,PTE)大鼠模型,观察线粒体及氧化应激反应相关指标的变化,并通过检测脑组织细胞凋亡及相关凋亡蛋白的表达,探讨创伤性癫痫下线粒体功能的变化并观察应用抗氧化剂α-硫辛酸(α-lipoic acid,α-LA)后各相关指标的改变,进一步探索神经细胞凋亡和超微结构损伤变化在创伤性癫痫的发病机制中的作用,以及抗氧化剂对这一病理重构过程的影响。方法:取45只雄性Wistar大鼠随机分成三组(对照组、创伤性癫痫组、α-硫辛酸干预组),每组15只。建立Fe Cl2所致颅脑创伤后癫痫大鼠模型,参考立体定位图谱埋藏不锈钢电极,记录各组大鼠脑电图(EEG)。采用尼氏染色法观察大鼠皮层及海马神经元情况,应用Western-Blot法检测与分析Caspase-3蛋白质,并观测线粒体中丙二醛(MDA)和一氧化氮(NO)的含量,检测Na+K+-ATP酶、Ca2+Mg2+-ATP酶、总ATP酶、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力及线粒体膜电位的变化,进一步了解神经元凋亡和超微结构损伤在PTE的发病机制。结果:1.正常对照组大鼠皮层电极脑电图(EEG)均未见丛集性棘波放电,创伤性癫痫组皮层电极EEG均描记到丛集性棘波放电,通过α-硫辛酸干预后,丛集性棘波放电现象明显改善。2.创伤性癫痫组大鼠出现癫痫发作,表现为(1)耳、面部痉挛性抽动,包括眨眼、动须、节律性咀嚼,双耳颤动等;(2)浑身颤动,节律性点头;(3)动物前肢阵发性抽搐,频率随时间增加;(4)双前肢抬起,呈半直立位,随后全身及四肢肌肉阵挛,不能直立,后倾跌倒;(5)动物四肢抽动,无法站立,出现全身强直阵挛发作。按Racine标准均达到中重度发作。发作结束后大鼠精神萎靡,活动及进食量减少。对照组大鼠则无癫痫发作,而α-硫辛酸干预组癫痫发作程度明显减轻。3.对各组大鼠大脑皮层与海马进行尼氏染色,创伤性癫痫组海马区锥体细胞的超微结构有明显改变,表现为细胞核发生变形、固缩,染色质固缩凝集在核膜的内表面,碎裂成碎片,在核膜附近形成多个密集的斑块,核膜不清晰;α-硫辛酸干预组神经元形态结构基本正常,核膜基本清晰,无染色质聚集;胞质内有少许水肿空白区。4.Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、总ATP酶活性在创伤性癫痫组与对照组比较均有显著的下降,而α-硫辛酸干预组则有明显的改善,两组比较有显著的统计学差异。线粒体细胞膜电位在癫痫组有明显下降,通过α-硫辛酸干预,能够抑制其下降。5.SOD与GSH-PX两指标在创伤性癫痫组浓度降低,MDA与NO两指标则升高,证实在癫痫发作时,氧化应激状态的存在。而α-硫辛酸干预组这些指标则有显著的改善,两者比较有明显的统计学差异。6.在创伤性癫痫组,细胞凋亡明显增多,凋亡相关蛋白Caspase-3表达明显上调,而α-硫辛酸干预组则细胞凋亡现象有显著的下降,同样Caspase-3表达水平也下降明显。结论:1.在Fe Cl2所致创伤性癫痫大鼠癫痫模型中,MDA、NO水平显著升高,Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、总ATP酶、SOD、GSH-Px活力则明显降低;提示在创伤性癫痫大鼠模型中的脑细胞线粒体内会产生大量的自由基,脑细胞发生氧化应激损伤,导致抗氧化防御功能明显下降;脑细胞线粒体中的能量供应受到相应的阻碍。2.α-硫辛酸对创伤性癫痫大鼠线粒体氧化应激损伤有保护作用。其机理可能与提高了脑组织中线粒体内抗氧化的相关酶的活性,使脑组织线粒体生物膜的脂质过氧化水平降低有关。3.在FeCl2所致创伤性癫痫大鼠模型中,氧化应激反应诱导神经细胞凋亡以及神经元超微结构的改变,α-硫辛酸保护的作用途径之一可能为抑制氧化应激诱导的神经细胞凋亡。
[Abstract]:Objective: epilepsy is a chronic nervous system disease, which is characterized by an excessive, stereotyped and abrupt abrupt characteristic. Traumatic brain injury (TBI) is one of the important causes of symptomatic epilepsy. The cerebral ischemia and hypoxia response caused by epileptic seizures causes the brain neurons to be damaged and produces a large number of free radicals, causing mitochondrial damage and line damage. In this study, the changes in mitochondrial and oxidative stress related indexes were observed by establishing the posttraumatic epilepsy (PTE) rat model, and the traumatic epilepsy was explored by detecting the apoptosis of the brain tissue and the expression of the associated apoptotic proteins. The changes in the function of the mitochondria and the changes of the related indexes after the application of antioxidant alpha -lipoic acid (alpha -LA) were observed. The effects of apoptosis and ultrastructural damage on the pathogenesis of traumatic epilepsy were further explored, and the effects of antioxidants on this pathological process. Methods: 45 males were taken. Sex Wistar rats were randomly divided into three groups (control group, traumatic epilepsy group, alpha lipoic acid intervention group) and 15 rats in each group. The model of epileptic rats after Fe Cl2 was established. The electroencephalogram (EEG) of each group was recorded in the stereotaxic map, and the neurons in the cortex and hippocampus of rats were observed by Nissl staining. Caspase-3 protein was detected and analyzed by Western-Blot, and the content of malondialdehyde (MDA) and nitric oxide (NO) in mitochondria was observed. The changes of Na+K+-ATP enzyme, Ca2+Mg2+-ATP enzyme, total ATP enzyme, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity and mitochondrial membrane potential were detected. The apoptosis and ultrastructure of neurons were further understood. Results: 1. the cortex electrode electroencephalogram (EEG) of the normal control group had no cluster spinous discharge, and the cortical electrode EEG of the traumatic epilepsy group was traced to the cluster spinous wave discharge. The cluster spinous discharge phenomenon obviously improved the epileptic seizures in the.2. traumatic epileptic group by the prognosis of the alpha lipoic acid. 1. (1) ears, facial spasmodic movement, including blinking, moving whiskers, rhythmic chewing, double ear tremor, (2) trembling, rhythmic nods, (3) the frequency of the forelimb spasms, frequency increased with time; (4) the double forelimbs were raised in a vertical position, with the posterior body and limbs muscle clonus, unable to erect, backward dip; (5) animal limbs twitching, (5) the animals' extremities twitching, unable to Standing, generalized tonic clonic seizures. Moderate and severe seizures were reached according to the Racine standard. After the end of the seizure, the rats were depressed and the activity and food intake decreased. The control group had no epileptic seizures, and the degree of epileptic seizures in the intervention group of alpha lipoic acid significantly alleviated the Nissl staining and traumatic epilepsy in the cerebral cortex and hippocampus of rats in each group. The ultrastructure of pyramidal cells in the hippocampus of the group was changed obviously, which showed that the nuclei were deformed and fixed, and the chromatin was condensed and condensed on the inner surface of the nuclear membrane, and the fragmentation became fragments. There were many dense patches near the nuclear membrane, and the nuclear membrane was not clear. The morphological structure of the neurons in the alpha lipoic acid intervention group was basically normal, and the nuclear membrane was basically clear and no chromatin was found. There was a significant decrease in the activity of.4.Na+-K+-ATP enzyme, Ca2+-Mg2+-ATP enzyme and total ATP enzyme in the traumatic epileptic group and the control group, but there was a significant improvement in the alpha lipoic acid intervention group. The two groups had significant statistical differences. The mitochondrial membrane potential decreased significantly in the epileptic group, through alpha. The intervention of lipoic acid could inhibit the decrease of the concentration of.5.SOD and GSH-PX two in the traumatic epileptic group, and the increase of MDA and NO two index, which confirmed the presence of oxidative stress during epileptic seizures, and these indexes were significantly improved in the alpha lipoic acid intervention group, and there was a significant statistical difference between the two groups,.6. in the traumatic epilepsy group, Apoptosis was significantly increased, apoptosis related protein Caspase-3 expression was obviously up-regulated, while alpha lipoic acid intervention group had a significant decrease in apoptosis, and the same level of Caspase-3 expression decreased obviously. Conclusion: 1. in the epileptic model of traumatic epileptic rats induced by Fe Cl2, the level of MDA, NO is significantly increased, Na+-K+-ATP enzyme, Ca2+-Mg2+-ATP enzyme, total A. The activity of TP, SOD and GSH-Px decreased significantly, suggesting that a large number of free radicals produced in the mitochondria of the brain cells in the rat model of traumatic epilepsy, the oxidative stress in the brain cells was damaged and the antioxidant defense function decreased obviously. The energy supply in the mitochondria of the brain cells was affected by the corresponding hindering.2. alpha lipoic acid to the traumatic epileptic rats Mitochondrial oxidative stress damage has protective effect. Its mechanism may improve the activity of mitochondrial antioxidant related enzymes in brain tissue, reduce the lipid peroxidation level of mitochondrial biofilm in brain tissue related to.3. in FeCl2 induced rat model of traumatic epilepsy, and induce neuronal apoptosis and neuronal ultrastructure induced by oxidative stress reaction. One of the protective pathways of alpha lipoic acid may be to inhibit oxidative stress induced neuronal apoptosis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1

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