盐酸法舒地尔对AD大鼠空间学习记忆能力的改善及其机制的研究
发布时间：2018-10-20 18:29
【摘要】：背景 阿尔茨海默病(Alzheimer's disease, AD)是老龄人群中最常见也是最重要的一种退变性神经疾病,临床上以进行性的认知功能障碍,特别是空间学习,记忆力障碍为主要特点。随人类预期寿命的增加,此种毁灭性疾病发病率逐年上升,已继心脏病,肿瘤,中风后成为人类疾病死亡的第四大病因。大量研究证明阿尔茨海默病的特征性病理改变与脑前叶和海马的老年斑沉积,神经纤维缠结和神经元凋亡紧密相连。但总体来说,阿尔茨海默病的发病机理仍不明确,且此种疾病目前尚无法彻底治愈。因此仍需要进行大量的研究。目前已有研究表明阿尔茨海默病的学习,记忆能力的进行性退化与突触功能障碍息息相关,而突触的结构和功能也被认为在病理上与认知功能障碍关系最为紧密。因此,突触功能障碍是改善认知功能障碍,延缓阿尔茨海默病发展的一个重要的治疗靶点。盐酸法舒地尔(Fasudil hydrochloride, FH)是法舒地尔的一种活性代谢产物。它是一个经典的Rho激酶(Rho kinase, ROCK)的拮抗剂。已有报道法舒地尔能改善老龄大鼠,脑缺血模型大鼠的认知功能障碍。然而,盐酸法舒地尔是否在阿尔茨海默病动物模型中同样具有抗痴呆作用仍不明了,而其抗痴呆作用的具体机制更鲜有研究。在本研究中,我们运用侧脑室注射链脲佐菌素(streptozotocin, STZ)的大鼠模型来观测盐酸法舒地尔对其学习,记忆能力的作用,以及其具体机制。 目的 本研究讨论了AD大鼠中空间学习,记忆能力缺损与海马突触改变的联系,以及通过检测STZ组大鼠学习,记忆能力,海马CA1区显微结构变化,突触囊泡素(synaptophysin, SYP)基因,蛋白表达和LIMK2, cofilin蛋白表达以及盐酸法舒地尔对上述指标的作用探讨盐酸法舒地尔抗痴呆作用的具体分子机制。本文为日后盐酸法舒地尔用于阿尔茨海默病的治疗提供的宝贵的理论基础。 方法 48只重250-300g的雄性SD大鼠由中南大学动物实验中心提供,并被随机分为4组(每组12只)：(1)假手术组(control);(2)盐酸法舒地尔对照组(sham+FH);(3)模型组(STZ)；(4)盐酸法舒地尔干预组(STZ+FH)。STZ组和STZ+FH组大鼠在第1天和第3天侧脑室注射STZ两次(1.5mg/kg体重),control组和sham+FH组大鼠给予等量的柠檬酸／柠檬酸钠缓冲液。Sham+FH和STZ+FH组大鼠腹腔注射盐酸法舒地尔(10mg/kg体重)4周,STZ组和control组大鼠则腹腔注射等量的生理盐水。学习和记忆能力用Morris水迷宫检测。HE染色和尼式染色检测海马CA1区神经元的形态。显微电镜观测海马CA1区突触显微结构。海马CA1区Gray I型突触界面参数用透射电镜观测及相关软件测量分析。real-time PCR和western blotting分别检测SYP基因和蛋白表达。LIMK2口cofilin的蛋白磷酸化水平也由western blotting检测得出。运用SPSS18.0软件对实验数据进行统计学分析。用均值±标准差的方式表示计量结果。以重复测量的两因素方差分析法统计分析Morris水迷宫中上台潜伏期,游泳速度,当总体方差分析有差异时,LSD法行组间两两比较分析组间差异。穿越平台次数用Wilcoxon signed-rank sum检验。其余变量用单因素方差分析,总体方差分析有差异时组间差异显著性用LSD行组间两两比较。P值0.05为有统计学意义。 结果 1.与control组相比,STZ组大鼠在第2天到第5天的上台潜伏期明显延长,表明学习能力受损；靶向限游泳时间和穿越平台次数明显下降,表明记忆能力受损。盐酸法舒地尔干预后,STZ+FH组大鼠中上述指标明显改善。Sham+FH组大鼠在Morris水迷宫检测中表现和control组大致相同,表明盐酸法舒地尔本身对假手术大鼠并无学习,记忆能力改善作用。 2.与control组相比,STZ组大鼠海马CA1区神经元数量减少且排列不规则,神经元细胞器显微结构明显受损。盐酸法舒地尔干预后,STZ+FH组大鼠海马CA1区神经元数量明显增加且排列较规则,神经元细胞器的显微结构破坏程度也有所改善。 3.与control组相比,STZ组大鼠海马SYP蛋白,基因表达明显下降,而STZ+FH组大鼠SYP基因,蛋白表达有所回升。 4.与control组相比,STZ组大鼠突触数量下降,伴有凹形突触,穿孔型突触比例下降。此外,还出现突触间隙变窄,PSD厚度变薄,突触活性带长度缩短,突触曲率降低改变。而盐酸法舒地尔干预后上述突触结构退行性改变均有所改善。 5.与control组相比,STZ组大鼠LIMK2和cofilin磷酸化水平有所上调。而盐酸法舒地尔能明显下调STZ+FH组大鼠LIMK2和cofilin的磷酸化水平。 结论 1.大鼠侧脑室注射STZ是一种阿尔茨海默病的动物模型。具有良好的可复制性和稳定性,是日后阿尔茨海默病研究中一种比较理想的AD动物模型。 2.STZ所致的海马神经元和突触结构异常可能是导致模型组大鼠认知功能障碍的原因之一。盐酸法舒地尔可能通过改善神经元和突触结构和功能起到改善STZ+FH组大鼠学习和记忆能力的作用。 3.盐酸法舒地尔所致的LIMK2和cofilin脱磷酸化过程可能是其保护突触结构和功能的分子机制之一。
[Abstract]:Background Alzheimer's disease (AD) is one of the most common and most important degenerative neurological diseases in the elderly population, which is mainly characterized by progressive cognitive dysfunction, especially spatial learning and memory disorder. Characteristic. With the increase of human life expectancy, the incidence of such destructive disease increases year by year, and has become the fourth major cause of human disease after heart disease, tumor and stroke. Etiology: A large number of studies have shown that the characteristic pathological changes of Alzheimer's disease are closely related to senile plaque deposits, nerve fiber entanglement and neuronal apoptosis in the anterior lobe and hippocampus of the brain. But in general, the pathogenesis of Alzheimer's disease is still unclear, and the disease is not yet complete It's cured. So there's still a lot to be done. At present, studies have shown that the study of Alzheimer's disease and the progressive degradation of memory ability are closely related to synaptic dysfunction, and the structure and function of synapses are also considered to be the most important in the pathogenesis of Alzheimer's disease Therefore, synaptic dysfunction is an important treatment for improving cognitive dysfunction and delaying the development of Alzheimer's disease. Target. Huudil hydrochloride (FH) is a kind of active metabolism in the method. Product. It is a classic Rho kinase (ROCK). Anti-agent. It has been reported that Shuisher can improve the cognitive function of aged rats and rats with cerebral ischemia model. However, it is unclear whether the anti-dementia effect is still unknown in the animal model of Alzheimer's disease, and the specific mechanism of anti-dementia effect is less. In this study, we used the rat model of streptozoocin (STZ) with lateral ventricular injection to observe the effect of hydrochloric acid method on its learning and memory ability, as well as its specific function. mechanism Objective: To study the relation between spatial learning and memory ability defect in AD rats and synaptic changes in hippocampus, and to study the changes in microstructure of hippocampal CA1 region, synaptophin (SYP) gene and protein by detecting the learning and memory ability of STZ rats. Expression of the expression and expression of LIMK2, cofilin protein and the effect of hydrochloric acid method on the above-mentioned indexes to explore the effect of hydrochloric acid method on anti-dementia Specific molecular mechanisms for use in the treatment of Alzheimer's disease. Bao's treasure Methods 48 male Sprague-Dawley rats weighing 250-300g were randomly divided into 4 groups (12 rats in each group): (1) sham operation group (control); (2) sham operation group (sham + F). H); (3) model group (STZ); (4) treatment group (STZ + FH). STZ group and STZ + FH group rats were injected STZ twice (1. 5mg/ kg body weight) on day 1 and day 3, control group and sham + FH group rats were given. An equal amount of citric acid/ sodium citrate buffer. Shams + FH and STZ + FH rats were injected into the abdominal cavity of rats for 4 weeks, STZ group and control group. Rats were injected intraperitoneally with equal amounts of physiological saline. Learning and memory Ability to detect with Morris water maze. HE staining and nintedanib Morphological and microscopic electron microscopic examination of neurons in hippocampal CA1 region Observation of synaptic microstructure in CA1 region of hippocampus of hippocampal CA1 region in hippocampal CA1 region Radio Mirror Observation and Related Software Measurement Analysis The expression of SYP gene and protein was detected. rn blotting detection results. SPSS18. 0 Statistical analysis of experimental data was performed by software. The method of variance analysis of two factors was used to analyze the incubation period and swimming speed in Morris water maze. Difference between the two comparative analysis groups between the two groups. Wilcoxon si was used for the number of crossing platforms. Received-Cadsum test. The remaining variables were analyzed by single-factor analysis of variance, and there was a difference in the overall variance of variance. Comparison between the two groups in the LSD line group .P Results 1. Compared with control group, the incubation period of STZ group in STZ group was significantly prolonged on day 2 to day 5, indicating impaired learning ability and limited swimming time. and the number of crossing platforms is obviously reduced, the memory capacity is impaired, After that, the above indexes in STZ + FH group were obviously improved. In the Morris water maze test, the rats were almost the same in the sham + FH group. The number of neurons in hippocampal CA1 region of STZ group was higher than that of control group. The number of neurons in hippocampal CA1 region of STZ + FH group was obviously increased and the arrangement was higher than that of group STZ + FH group. Compared with control group, the expression of SYP protein and gene in hippocampus of STZ group was obviously lower than that of control group. Compared with control group, the SYP gene and protein expression of STZ + FH group increased. The number of synapses in the STZ group decreased, accompanied by a decrease in the synapse of the concave synapse and the perforation type. In addition, synaptic cleft also appeared. Narnarrowed, reduced PSD thickness, reduced synaptic activity band length, reduced synaptic curvature The changes of the postsynaptic structure were improved after the intervention of the hydrochloric acid method, and the changes of the postsynaptic structure were improved. Compared with the group, the phosphorylation level of LIMK2 and cofilin in STZ group was up-regulated. Obvious Down-regulation of LIMK2 and cofilin in STZ + FH Rats Conclusion 1. Intracerebroventricular injection of STZ in rats is an animal model of Alzheimer's disease. There is good replicability and stability, which is an ideal AD animal model in the study of Alzheimer's disease. 2. The abnormality of hippocampal neurons and synaptic structures caused by STZ may be one of the causes of cognitive dysfunction in the model group. It is possible to improve the learning and memory ability of STZ + FH rats by improving the structure and function of neurons and synapses.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16
[Abstract]:Background Alzheimer's disease (AD) is one of the most common and most important degenerative neurological diseases in the elderly population, which is mainly characterized by progressive cognitive dysfunction, especially spatial learning and memory disorder. Characteristic. With the increase of human life expectancy, the incidence of such destructive disease increases year by year, and has become the fourth major cause of human disease after heart disease, tumor and stroke. Etiology: A large number of studies have shown that the characteristic pathological changes of Alzheimer's disease are closely related to senile plaque deposits, nerve fiber entanglement and neuronal apoptosis in the anterior lobe and hippocampus of the brain. But in general, the pathogenesis of Alzheimer's disease is still unclear, and the disease is not yet complete It's cured. So there's still a lot to be done. At present, studies have shown that the study of Alzheimer's disease and the progressive degradation of memory ability are closely related to synaptic dysfunction, and the structure and function of synapses are also considered to be the most important in the pathogenesis of Alzheimer's disease Therefore, synaptic dysfunction is an important treatment for improving cognitive dysfunction and delaying the development of Alzheimer's disease. Target. Huudil hydrochloride (FH) is a kind of active metabolism in the method. Product. It is a classic Rho kinase (ROCK). Anti-agent. It has been reported that Shuisher can improve the cognitive function of aged rats and rats with cerebral ischemia model. However, it is unclear whether the anti-dementia effect is still unknown in the animal model of Alzheimer's disease, and the specific mechanism of anti-dementia effect is less. In this study, we used the rat model of streptozoocin (STZ) with lateral ventricular injection to observe the effect of hydrochloric acid method on its learning and memory ability, as well as its specific function. mechanism Objective: To study the relation between spatial learning and memory ability defect in AD rats and synaptic changes in hippocampus, and to study the changes in microstructure of hippocampal CA1 region, synaptophin (SYP) gene and protein by detecting the learning and memory ability of STZ rats. Expression of the expression and expression of LIMK2, cofilin protein and the effect of hydrochloric acid method on the above-mentioned indexes to explore the effect of hydrochloric acid method on anti-dementia Specific molecular mechanisms for use in the treatment of Alzheimer's disease. Bao's treasure Methods 48 male Sprague-Dawley rats weighing 250-300g were randomly divided into 4 groups (12 rats in each group): (1) sham operation group (control); (2) sham operation group (sham + F). H); (3) model group (STZ); (4) treatment group (STZ + FH). STZ group and STZ + FH group rats were injected STZ twice (1. 5mg/ kg body weight) on day 1 and day 3, control group and sham + FH group rats were given. An equal amount of citric acid/ sodium citrate buffer. Shams + FH and STZ + FH rats were injected into the abdominal cavity of rats for 4 weeks, STZ group and control group. Rats were injected intraperitoneally with equal amounts of physiological saline. Learning and memory Ability to detect with Morris water maze. HE staining and nintedanib Morphological and microscopic electron microscopic examination of neurons in hippocampal CA1 region Observation of synaptic microstructure in CA1 region of hippocampus of hippocampal CA1 region in hippocampal CA1 region Radio Mirror Observation and Related Software Measurement Analysis The expression of SYP gene and protein was detected. rn blotting detection results. SPSS18. 0 Statistical analysis of experimental data was performed by software. The method of variance analysis of two factors was used to analyze the incubation period and swimming speed in Morris water maze. Difference between the two comparative analysis groups between the two groups. Wilcoxon si was used for the number of crossing platforms. Received-Cadsum test. The remaining variables were analyzed by single-factor analysis of variance, and there was a difference in the overall variance of variance. Comparison between the two groups in the LSD line group .P Results 1. Compared with control group, the incubation period of STZ group in STZ group was significantly prolonged on day 2 to day 5, indicating impaired learning ability and limited swimming time. and the number of crossing platforms is obviously reduced, the memory capacity is impaired, After that, the above indexes in STZ + FH group were obviously improved. In the Morris water maze test, the rats were almost the same in the sham + FH group. The number of neurons in hippocampal CA1 region of STZ group was higher than that of control group. The number of neurons in hippocampal CA1 region of STZ + FH group was obviously increased and the arrangement was higher than that of group STZ + FH group. Compared with control group, the expression of SYP protein and gene in hippocampus of STZ group was obviously lower than that of control group. Compared with control group, the SYP gene and protein expression of STZ + FH group increased. The number of synapses in the STZ group decreased, accompanied by a decrease in the synapse of the concave synapse and the perforation type. In addition, synaptic cleft also appeared. Narnarrowed, reduced PSD thickness, reduced synaptic activity band length, reduced synaptic curvature The changes of the postsynaptic structure were improved after the intervention of the hydrochloric acid method, and the changes of the postsynaptic structure were improved. Compared with the group, the phosphorylation level of LIMK2 and cofilin in STZ group was up-regulated. Obvious Down-regulation of LIMK2 and cofilin in STZ + FH Rats Conclusion 1. Intracerebroventricular injection of STZ in rats is an animal model of Alzheimer's disease. There is good replicability and stability, which is an ideal AD animal model in the study of Alzheimer's disease. 2. The abnormality of hippocampal neurons and synaptic structures caused by STZ may be one of the causes of cognitive dysfunction in the model group. It is possible to improve the learning and memory ability of STZ + FH rats by improving the structure and function of neurons and synapses.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16
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