Nrf2-ARE通路对戊四氮点燃小鼠癫痫模型的脑保护作用及其机制研究

发布时间：2018-04-22 06:30

  本文选题：癫痫 + Nrf2　； 参考：《河北医科大学》2014年硕士论文

【摘要】：目的：癫痫是多种病因引起的慢性脑部疾病,通过引起大脑神经元反复地、过度地超同步化放电，导致中枢神经系统功能短暂性失常的临床表现。近年的研究结果表明,人体内过多的自由基(Free Radical, FR)积累与许多慢性疾病都与有关。氧自由基导致的氧化应激损伤目前已被广泛研究证实为癫痫发病的重要机制之一。已经有研究证明，无论是癫痫动物实验模型还是癫痫患者的脑内均存在着活跃的自由基反应，癫痫发作时自由基进一步明显增加，远远超过机体对自由基的清除能力，导致神经元和线粒体的损伤,最终可加重癫痫的发生。清除氧自由基、抗氧化的治疗方法能为癫痫的药物治疗提供新的靶点。 转录因子NF-E2相关因子(NF-E2-related factor2，Nrf2)是细胞氧化应激反应中的关键因子，Nrf2通过与抗氧化反应元件（antioxidant responseelement，ARE）相互作用来调节抗氧化蛋白和Ⅱ相解毒酶的表达。既往研究证实，Nrf2-ARE通路对神经系统疾病如：肌萎缩侧索硬化症(ALS)、帕金森病、脑出血、脑梗死和脑外伤等，具有很强的神经保护功能，但是其在癫痫发病中抗氧化应激损伤的相关报道甚少，本实验主要研究戊四氮（pentylenetetrazoloe，PTZ）点燃慢性癫痫小鼠脑内Nrf2-ARE通路的保护作用。使用基因型为Nrf2(+)和基因型为Nrf2(-)的两种小鼠制作戊四氮（pentylenetetrazoloe，PTZ）点燃慢性癫痫小鼠模型，观察Nrf2-ARE通路阻断后戊四氮致痫小鼠痫性发作等级、小鼠海马CA1区神经细胞损伤情况及海马组织中Nrf-ARE通路表达产物血红素氧合酶-1(heme oxygenase1，HO-1)表达量的变化情况，评价Nrf2-ARE通路对癫痫过程中神经细胞的保护作用并对其机制进行初步探讨，为阐明癫痫的病理生理机制提供实验依据，同时为癫痫治疗提供新的靶点，为抗癫痫药物的选择提供新的思路。 方法：选择实验动物并分组：选择健康成年雄性C57/B小鼠，基因型为Nrf2(+)、经PCR鉴定基因型为（Nrf2-/-）的成年雄性Nrf2基因敲除（Nrf2-KO）小鼠，由河北医科大学动物实验中心及省二院神经内科实验室提供，适应性饲养1周后，随机分成四组：C57小鼠对照组（C-Control）n=30、C57小鼠致痫组（C-PTZ）n=30、Nrf2基因敲除小鼠对照组（N-Control）n=30、Nrf2基因敲除小鼠致痫组（N-PTZ）n=30。 模型的制备：癫痫组采用戊四氮慢速点燃癫痫模型：腹腔注射给药：隔天给药，26天内给药13次，每次均为上午9:00-11:30进行。给药前称体重，致痫组每只小鼠腹腔注射0.5%的PTZ，剂量为32mg/kg，，合0.0064ml/g，对照组按照0.0064ml/g给予生理盐水腹腔注射。注射后将小鼠放至透明箱中观察30min，记录小鼠癫痫发作的最大级别。痫性发作的发作等级变化按照Racine六级评价标准：0级，无任何反应；Ⅰ级，湿狗样抖动、面部抽搐及咀嚼；Ⅱ级，颈部肌肉痉挛表现为点头和/或甩尾；Ⅲ级，一侧前肢阵挛；Ⅳ级，双侧前肢阵挛伴立；Ⅴ级，全身阵挛，失去平衡，跌倒。戊四氮点燃过程：采用慢点燃法，隔天给予小鼠PTZ。连续三次记录到4~5级点燃即为点燃成功，纳入实验。造模成功后继续隔日给予戊四氮刺激一次，诱导癫痫发作。制模过程中小鼠出现死亡或不符合模型要求者，予以剔除并随机补充，保证每组数量30只。 取材：最后一次PTZ刺激结束48小时后，每组随机选取5只小鼠用于尼氏染色技术，麻醉后用4%的多聚甲醛液灌流固定后，取小鼠海马CA1区域的脑组织标本制作成蜡块，进行尼氏染色，测定小鼠海马CA1区神经细胞数；每组随机选取5只小鼠麻醉后用1%多聚甲醛-1.25%戊二醛0.1M磷酸缓冲液灌流固定后，取小鼠海马区域的脑组织标本，经透射电子显微镜观察海马组织CA1区神经元亚细胞结构；每组取20只小鼠麻醉后快速在冰块上提取小鼠海马组织，利用western blot技术半定量测定HO-1的含量。 结果： 1四组小鼠在痫性发作等级方面的比较：N-PTZ组比C-PTZ组的平均痫性发作等级显著升高（P0.05）；N-Control组与C-Control组均无痫性发作。 2四组小鼠脑组织尼氏染色海马CA1区神经元细胞数目的比较：致痫组均比空白对照组的神经元数目明显减少（P0.05）；N-PTZ组比C-PTZ组的神经元数目显著减少（P0.05）；N-Control组与C-Control组差异无统计学意义（P0.05）。 3四组小鼠脑组织海马神经元透射电镜观察结果比较：电镜观察示，空白对照组，即C-control组及N-control组：海马CA1区细胞大体结构未发现异常。细胞内神经元核膜清晰，胞质中细胞器丰富，胞质内可见线粒体、粗面内质网和大量的多核糖体。N-PTZ组：海马CA1区神经元细胞可见明显的大体结构损伤，出现细胞体的破碎。细胞内核膜内陷或边界不清，核质不均匀，异染色质积聚成块、增多、边集，细胞质中线粒体出现肿胀、线粒体嵴发生断裂甚至空化，粗面内质网池扩张、附着的核糖体脱落，游离核糖体解聚，重者出现胞浆空化。C-PTZ组：海马CA1区有部分神经元受损，且受损程度较N-PTZ组明显较轻，表现为核膜边界不清，细胞变形，电子密度影呈水肿表现，少数细胞质浓缩，粗面内质网排列紊乱，多聚核糖体稍减少，部分线粒体有嵴断裂现象。 4四组小鼠海马区脑组织HO-1的表达western blot半定量比较：N-control组比C-control组表达量减少（P0.05）；N-PTZ组比C-PTZ组HO-1表达量明显减少（P0.05）；N-PTZ组比N-control组HO-1表达量明显增多（P0.05）；C-PTZ组比C-control组HO-1表达量明显增多（P0.05）。 结论： 戊四氮点燃的慢性癫痫模型能够很好模拟了人类海马神经元的损伤。Nrf2基因缺陷小鼠的平均癫痫发作等级更高，且其海马区神经元在癫痫病程中受到更大的损伤，与此同时海马组织中血红素氧合酶-1的含量明显降低。这可能表示癫痫癫痫病程中，Nrf2基因可能对癫痫小鼠的海马神经元有保护作用，且可能是通过激活Nrf2-ARE通路进而增加脑组织中HO-1的表达来实现的。
[Abstract]:Objective: epilepsy is a chronic brain disease caused by a variety of causes, which results in a clinical manifestation of transient dysfunction of the central nervous system by overly oversynchronous discharge caused by the recurrent brain neurons. Recent results show that the accumulation of Free Radical (FR) in the human body is associated with many chronic diseases. Oxidative stress damage caused by oxygen free radicals has been widely studied as one of the important mechanisms of epilepsy. Research has shown that there is an active free radical reaction in both epileptic animal model and epileptic patients, and the free radicals in epileptic seizures are further increased, far more than the body's freedom. The scavenging ability of the base leads to the damage of neurons and mitochondria, which can eventually aggravate the occurrence of epilepsy. Scavenging oxygen free radicals and antioxidation therapy can provide new targets for the treatment of epilepsy.
The transcription factor NF-E2 related factor (NF-E2-related FACTOR2, Nrf2) is a key factor in cellular oxidative stress. Nrf2 regulates the expression of antioxidant protein and phase II detoxification enzyme by interacting with the antioxidant response element (antioxidant responseelement, ARE). Previous studies have confirmed that the Nrf2-ARE pathway is a disease of the nervous system, such as the muscle. Amyotrophic lateral sclerosis (ALS), Parkinson's disease, cerebral hemorrhage, cerebral infarction and brain injury have strong neuroprotective functions. However, there are few reports on the damage of antioxidant stress in epilepsy. This experiment mainly studies the protective effect of pentylenetetrazoloe (PTZ) on the protection of Nrf2-ARE pathway in the brain of chronic epileptic mice. Two mice with genotype Nrf2 (+) and genotype Nrf2 (-) were used to make amyl four nitrogen (pentylenetetrazoloe, PTZ) to kindle chronic epilepsy in mice. The level of epileptic seizures in epileptic mice induced by Nrf2-ARE pathway, the damage of neurons in CA1 region of hippocampus and the heme oxygenation of the expression products of Nrf-ARE pathway in hippocampus of hippocampus were observed. The change of the expression of enzyme -1 (heme oxygenase1, HO-1), to evaluate the protective effect of Nrf2-ARE pathway to the neural cells in the process of epilepsy and to discuss its mechanism, provide experimental basis for elucidating the pathophysiological mechanism of epilepsy, and provide new targets for the treatment of epilepsy, and provide a new idea for the selection of antiepileptic drugs.
Methods: select experimental animals and group: select healthy adult male C57/B mice and genotype Nrf2 (+). The adult male Nrf2 gene knockout (Nrf2-KO) mice were identified by PCR (Nrf2-/-), which were provided by the animal experiment center of Hebei Medical University and the second hospital of the province. After 1 weeks of adaptive feeding, they were randomly divided into four groups: C 57 mice control group (C-Control) n=30, C57 mice induced epilepsy group (C-PTZ) n=30, Nrf2 gene knockout mice control group (N-Control) n=30, Nrf2 knockout mice induced seizure group (N-PTZ) n=30.
Model preparation: epileptic group was treated with an amyl four nitrogen slow kindling model of epilepsy: intraperitoneal injection: medication every other day and 13 times in 26 days, each time was 9:00-11:30 a.m., before the drug was given weight, each mice in the epilepsy group were injected with 0.5% PTZ, the dose was 32mg/kg, and 0.0064ml/g, and the control group was given the saline abdomen according to 0.0064ml/g. The mice were injected into the cavity. The mice were placed in the transparent box to observe 30min and record the maximum level of epileptic seizures in mice. The level of seizures was changed according to the standard of Racine six: grade 0, without any reaction; grade I, wet dog jitter, facial convulsion and chewing; class II, neck muscle spasms were nodding and / or tail flick; class III, One side of the forelimb clonus; grade IV, bilateral forelimb clonus and clonus; grade V, general clonus, loss of balance, fall. The amyl four nitrogen kindling process: using the slow ignition method, the mice were given three consecutive records to the PTZ. kindling every other day to kindle successfully, which was brought into the experiment. After the success of the model, the stimulation of the amyl four nitrogen was given and the epileptic seizures were induced. The mice died during the mold making process or did not meet the requirements of the model. They were eliminated and randomly supplemented to ensure that there were 30 in each group.
48 hours after the final PTZ stimulation, 5 mice in each group were randomly selected for Nissl staining. After anesthesia, the brain tissue samples from the CA1 region of the hippocampus of the mice were made into wax blocks, and Nissl staining was performed to determine the number of neurons in the CA1 region of the hippocampus of mice. 5 mice were randomly selected for each group. After drunken injection of 1% polyformaldehyde -1.25% glutaraldehyde 0.1M phosphate buffer, the brain tissue specimens of the hippocampus region of mice were collected, and the subcellular structure of neurons in the hippocampal CA1 region was observed by transmission electron microscope. 20 mice in each group were extracted and extracted quickly on the ice block. Semi quantitative determination by Western blot technique was used. The content of HO-1.
Result锛

本文编号：1786027