节点文献
督脉不同电针对拟AD模型-SAMP8小鼠额叶Aβ代谢途径的影响
【作者】 许安萍；
【导师】 李志刚；
【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2014， 博士
【摘要】 1研究目的AD是临床痴呆最主要的类型,以进行性神经变性为特征,在疾病的某一阶段还常伴有明显的精神行为异常,严重损伤患者的生活能力和社会功能,同时给家庭和社会亦带来沉重负担,已成为当今老年医学面临的最为严峻的问题之一。综合以往研究,关于AD病理机制存在多种假说,每种假说都与AD大脑一些重要生物化学成分改变相关,特别是涉及重要蛋白质和酶等生物大分子的改变。其中,β-淀粉样蛋白(Ap)假说得到广泛认可,该假说认为Aβ是AD发病的关键环节,其毒性引发的连锁反应是AD一系列病理改变的基础。因此,有效抑制Aβ分泌,干扰Ap毒性引发的连锁反应,打断其恶性循环是治疗AD的一个重要策略。依据导师李志刚教授长期临床经验并且结合前期动物实验研究发现,通督启神针法能够促进神经细胞修复再生,有效改善脊髓损伤、颅脑损伤等与中枢神经系统相关疾病,是一种实用性较强的治疗方法。临床AD具有心理学特性,以往将传统针刺疗法与音乐疗法结合治疗AD的研究尚不多见。本研究以与AD发病密切相关的Aβ蛋白为切入点,首次将音乐电针疗法作为一种干预手段引入AD的治疗中,采用神经行为学、分子生物学等方法,深入探讨通督启神针法指导下的不同电针对AD模型小鼠额叶皮层Aβ蛋白生成途径和清除途径两方面的影响,同时对比分析脉冲电针与音乐电针的疗效差异,以期为临床应用音乐电针治疗AD以及其他脑相关疾病提供科学实验依据。2研究方法本研究采用动物实验方法,以SAMR1和SAMP8小鼠作为研究对象,分为空白对照组(B组)、模型对照组(C组)、药物对照组(D组)、音乐对照组(M组)、音乐电针组(ME组)和脉冲电针组(PE组)6个组别,干预15天后,采用Morris水迷宫实验评估各组小鼠行为学的变化,比较不同电针对学习记忆能力的影响；采用尼氏染色观察各组小鼠额叶皮层神经细胞的病理改变,比较不同电针对细胞形态学的影响；采用免疫组织化学方法和Western Blot方法检测各组小鼠额叶皮层Aβ40和BACE1含量,并应用Real time-PCR法检测BACE1mRNA表达情况,比较不同电针对Aβ生成途径的影响；采用免疫组织化学方法和Western Blot方法检测各组小鼠额叶皮层Aβ降解酶IDE、 NEP以及相关转运受体LRP1、RAGE的表达情况,比较不同电针对Aβ清除途径的影响。3研究结果3.1定位航行实验中,与B组比较,C组小鼠逃避潜伏期基线较高,而且经过5天训练并没有明显缩短,同时游泳总路程基线较低,并且有持续下降趋势。D组、ME组和PE组逃避潜伏期随训练时间增加呈波动性下降趋势,以ME组尤为突出,其出现明显变化时间最早,随后出现明显变化的是D组、PE组,而M组在5天的训练中变化并不明显。在游泳总路程上ME组与C组比较,具有显著性差异,且在某些时间点上优于D组和PE组。在空间探索实验中,与B组比较,C组原平台象限停留时间及游泳路程显著下降,搜索策略多呈随机式,无明显倾向性,而D组、ME组和PE组与C组比较有明显差异,搜索多停留在原平台所在象限或附近,ME组与D组无显著性差异,略优于PE组。3.2B组额叶皮层的神经元体积较大,形态规则,胞浆内布满尼氏体,呈深蓝色。C组额叶皮层的神经元体积较小,形态不规则,有较多细胞失去完整形态,神经元胞浆内尼氏体着色浅淡,有一定程度的脱失。经不同电针及药物干预的治疗组额叶皮层的神经元体积大于C组,但明显小于B组,形态较规则,胞浆内尼氏体有一定程度的恢复。M组较C组形态学无显著性变化。3.3与B组比较,C组Aβ40表达明显升高,差异具有显著性。与C组比较,D组、ME组、PE组Aβ40表达水平均明显下降,但M组无显著性变化。与D组比较,ME组Aβ40表达水平无明显差异,PE组和M组仍明显增高。ME组Aβ40水平与PE组无显著差异。3.4与B组比较,C组小鼠额叶皮层BACE1mRNA相对表达量及BACE1表达水平明显升高。与C组比较,D组、ME组、PE组两指标均有不同程度下调,差异具有显著性,其中ME组与D组作用相当,且略强于PE组,而M组无明显变化。3.5与B组比较,C组IDE、NEP含量均明显降低,差异具有显著性。与C组比较,D组、ME组、PE组两指标表达水均有不同程度提高。在提高IDE表达方面,ME组优于D组和PE组,而在改善NEP表达方面,ME组与PE组无显著性差异,且稍逊于D组。M组两指标的变化无统计学意义。3.6与B组比较,C组LRP1表达水平明显下降,而RAGE明显升高,差异具有显著性。与C组比较,ME组和PE组两指标的表达水平均明显改善,差异具有统计学意义,且ME优于PE,而D组和M组在两个指标的表达上与C组相比无显著性差异。4研究结论4.1通督启神针法指导下的电针疗法对拟AD模型——SAMP8小鼠学习记忆障碍具有改善作用。4.2通督启神针法指导下的电针疗法可修复AD模型——SAMP8小鼠额叶皮层神经细胞的损伤,对神经细胞具有一定的保护作用,并且可以通过纠正Ap代谢障碍,降低Aβ40的表达水平,从而发挥治疗痴呆的作用。4.3通督启神针法指导下的电针疗法发挥纠正Aβ代谢障碍的作用,通过两条途径实现：第一,干预Aβ生成途径。电针通过抑制BACE1表达,减少APP通过淀粉样蛋白源性加工途径产生Aβ；第二,干预Ap清除途径。电针通过增加IDE、NEP等Ap降解酶表达,双向调节Ap蛋白相关转运体LRP1及RAGE水平,促进脑内Aβ降解及转运。4.4音乐电针和脉冲电针均可通过改善神经细胞损伤和纠正Ap代谢障碍,从而促进拟AD模型——SAMP8小鼠学习记忆能力恢复。虽然在下调Aβ40和保护神经细胞方面,音乐电针相对于脉冲电针的优势没有统计学意义,但是在改善行为学及大部分Aβ相关降解酶和转运受体方面,音乐电针的优越性得以体现。综合行为学结果及与Ap代谢相关的分子生物学指标,音乐电针疗法的作用最全面。
【Abstract】 1ObjectveAlzheimer’s disease (AD) is the most common type of clinical dementia, characterized by progressive neurodegenerative, and often accompanied by significant mental and behavioral abnormalities in the certain stage. It seriously affects the life ability and social function of the patient, at the same time also brings heavy burden to family and society, which has become one of the most serious problems facing geriatric medicine today. Comprehensive previous research, there are many hypotheses about AD pathological mechanism, each hypothesis associated with some important biological chemical composition in brain, especially the important biological macromolecules such as protein and enzymes involved in the biochemistry change. At present, β-amyloid(Aβ)pothesis is widely recognized, which points that AP is the key to the pathogenesis of AD, and the chain reaction caused by its toxicity is the basis of pathological changes in AD. Therefore, an important strategy for the treatment of AD is interrupting the vicious cycle caused by Aβ,by means of restraining Aβ secretion and deposition and disturbing the chain reaction caused by its toxicity in the multi-link and multi-path effectively.Professor Li Zhigang based long-term clinical experience combined with previous animal experiments summarized Dredging Governor Meridian and Awakening Mind Method. It is a kind of practical treatment, which can effectively improve the diseases associated with central nervous system by promoting nerve cell regeneration, such as spinal cord injuries and traumatic brain injury. AD has a characteristic of psychology, yet the research about combining the traditional acupuncture and music therapy in the treatment of AD is still rare in the past. This study is based on β-amyloid hypothesis, with Aβ as the breakthrough point, at the same time including music therapy as an intervention to the treatment of AD. We will delve into different electro-acupuncture treatment guided by Dredging Governor Meridian and Awakening Mind Method for Aβ production means and clear way in frontal cortex of AD model, as well as Compare the curative effect of pulse electro-acupuncture and music electro-acupuncture differences, in order to provide a scientific basis for its clinical application of music electro-acupuncture treatment of AD and other brain-related diseases.2MethodsIn this study, SAMR1and SAMP8mice were used as the research object. They were randomly divided into blank control group (B group), model control group(C group), drug control group(D group), music control group(M group), pulse electro-acupuncture group (PE group) and music electro-acupuncture group(ME group). Morris Water Maze Test was used to detected the learning memory ability of mice, and then compared the influence of two kinds of electro-acupuncture therapy for the ability of learning and memory; Nissl staining was used to observe the pathological changes of mice frontal cortex neurons, and then compared the effect of different therapy; Immunohistochemistry and western blot were used to assay the expression levels of Aβ40and BACE1in frontal cortex, as well as Real-time PCR was used to measure the expression of BACElmRNA, so as to compare the effect of different therapy for Aβ generation; Immunohistochemistry and western blot were used to assay the expression levels of IDE, NEP, LRP1and RAGE in frontal cortex, so as to compare the effect of different therapy for Aβ clearance.3Results3.1Compared with B group, escape latency period baseline was higher and it was not significantly reduced after five days training, as well as total distance of swimming baseline was lower and continued to drop in C group. Escape latency period in groups of D, ME and PE shortened with the increase of training time, especially ME group, but it did not change significantly in M group. Total distance of swimming in ME group was significantly longer than in C group, also longer than D and PE group in some points. In the experiment of space exploration, Search strategy in C group was mostly random and with no obvious bias, as well as the original platform quadrant residence time and swimming distance dropped significantly compared with B group; D, ME and PE groups were better than C group, and ME group was the same as D group but a litter better than PE group.3.2Nissl staining showed frontal cortex pyramidal cells in B group arranged closely and orderly. But in C group, the pyramidal cells in frontal cortex was disorganized or wrinkled like, even incomplete form. Moreover, the number decreased significantly. In addition to the M group, the situation in groups of D, ME and PE were better than in C group.3.3Compared with B group, Aβ40expression level of frontal cortex in C group was significantly increased. Compared with C group, it was decreased obviously in groups of D, ME and PE except M group. Compared with D group, ME group was no significant difference, but PE and M groups were still significantly higher than it. There was no significant difference in Aβ40levels between ME group and PE group.3.4Compared with B group, BACE1mRNA relative expression and BACE1expression levels of frontal cortex in C group were significantly increased. Compared with C group, both in groups of D, ME and PE had different degrees of reduction and the difference was significant. ME group was the same as D group but a litter better than PE group.3.5Compared with B group, expression level of IDE and NEP in frontal cortex in C group was significantly decreased. Compared with C group, both in groups of D, ME and PE had a certain degree of improvement. ME group was better than D and PE groups in terms of regulating the expression of IDE, and there was no significant difference in regulating the expression of NEP between ME group and PE group, but both were not as good as D group. Both IDE and NEP in M group were not statistically significant.3.6Compared with B group, expression level of LRP1in frontal cortex in C group was significantly decreased, but RAGE significantly increased. Compared with C group, both in ME group and PE group were significantly improved, and ME group better than PE group. However, both LRP1and RAGE in groups of D and M were No obvious change.4Conclusions4.1Both pulse electro-acupuncture and music electro-acupuncture treatment guided by Dredging Governor Meridian and Awakening Mind Method can improve the learning and memory disorders of S AMP8mice with AD feature.4.2Both pulse electro-acupuncture and music electro-acupuncture treatment guided by Dredging Governor Meridian and Awakening Mind Method can repair the damage to the frontal cortex neurons of SAMP8mice with AD feature, thus play a role in protecting neurons. In addition, they can reduce the expression levels of Aβ40and correct Aβ metabolic disorder. So they have a role in the treatment of AD.4.3Electro-acupuncture treatment guided by Dredging Governor Meridian and Awakening Mind Method play a role in correcting A(3metabolic disorders through two ways. On the one hand, it can restrain the generation of A(3by reducing the expression levels of BACE1;on the other hand, it can promote the clearance of Aβ by increasing the expression levels of IDE and NEP which belong to Aβ degrading enzyme and regulate the content of LRP1and RAGE which belong to A(3protein related transporters.4.4By means of repairing the damage to neurons and correcting Aβ metabolic disorders in the frontal cortex, both pulse electro-acupuncture and music electro-acupuncture treatment guided by Dredging Governor Meridian and Awakening Mind Method can improve the learning and memory disorders of SAMP8mice with AD feature. There is no difference between the two treatments in adjusting individual indicators, but music electro-acupuncture treatment has more advantage on the whole.