益智汤对APP695转基因小鼠的治疗作用及其机理研究

【作者】 张忠；

【导师】 白丽敏；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 阿尔茨海默病(AD)是一种常见的中枢神经系统退行性疾病,其主要病理特征为脑内β-淀粉样蛋白(Aβ)沉积形成老年斑(SP)和神经原纤维缠结(NFT),并伴有突触和神经元丢失等,严重损害认知功能,造成患者生活自理能力障碍和精神行为异常。研究表明老年斑的主要成分是Aβ, Aβ具有神经毒性,其在脑实质内沉积是AD发病的关键病理机制。中医理论认为AD发病由于肾虚髓亏,痰浊阻窍,故多用补肾填髓、化浊开窍法来治疗AD。益智方由淫羊藿、西洋参、枸杞、石菖蒲、天麻、川芎等6味药组成,方中淫羊藿、西洋参、枸杞补肾填髓；天麻、石菖蒲、川芎化浊开窍。本实验通过益智汤对APP695转基因小鼠的治疗作用环节及其机理研究,揭示补肾填髓、化浊开窍法治疗AD的作用机制,为中医临床治疗AD提供实验依据和增加新的内涵。本实验分为两部分：第一部分为整体实验,以13月龄APP695转基因小鼠为动物模型,随机分为模型组、益智汤治疗组,同月龄未经转基因处理的C57BL/6J小鼠做为正常对照组。益智汤组小鼠每天根据体重以益智汤(1ml/100g)灌胃,模型组和正常组给予等体积蒸馏水灌胃。益智汤治疗3个月后：(1)使用电迷宫、水迷宫检测各组小鼠学习记忆能力；(2)使用免疫组化技术和计算机图像分析技术测定各组小鼠大脑皮层、海马APP、Aβ1-40、Aβ1-42、Caspase3的表达情况；(3)应用ELISA技术定量测定各组小鼠脑组织和脑脊液Aβ1-42的浓度；(4)使用光镜和电镜观察各组小鼠脑组织的一般病理变化和超微结构病变；(5)末端转移酶介导的原位缺口末端标记染色(Tune1)法观察各组小鼠脑组织神经细胞凋亡情况。第二部分为离体实验,选取体外培养8天的胎鼠大脑皮质神经元,加入不同浓度已经老化处理的Aβ25-35(5、10、20及40μmol/L)作用后,MTT比色法和LDH漏出量检测各组神经细胞活力和细胞膜功能的影响,倒置显微镜下观察神经元形态变化,来选择合适浓度老化处理的Aβ25-35复制AD细胞模型。模型组细胞加入益智汤含药大鼠血清,选择相同的观察指标,观察益智汤大鼠血清对Aβ25-35损伤神经元的保护作用。主要实验结果如下：1.益智汤可改善APP695转基因小鼠行为学习和记忆能力Y型电迷宫显示模型组需要学习达标次数明显多于正常组,且记忆达标次数明显低于正常组(P＜0.01)。中药组所需达标次数少于模型组(P＜0.05),记忆能力明显改善(P＜0.01)。水迷宫结果显示模型组和中药组潜伏期多于正常组,进入盲端次数也多于正常组,与正常组有显著差异(P＜0.01,)。与模型组相比,中药组的潜伏期缩短,且进入盲端次数减少(P＜0.05)。说明16月龄APP695转基因小鼠学习记忆能力比正常对照组明显下降,较好地模拟了AD的行为学特征。中药益智汤能改善APP转基因模型小鼠的学习和记忆能力。2.益智汤可降低APP695转基因小鼠脑组织内APP、Aβ1-40和Aβ1-42的表达免疫组化结果显示模型组皮质和海马CA1区APP、Aβ1-40和Aβ1-42阳性神经细胞较正常组明显增加,且染色-加深,二者相比有显著差异。与模型组相比,中药组小鼠皮质和海马CA1区APP、Aβ1-40和Aβ1-42的阳性细胞较少,且着色变浅,二者相比有显著差异。ELISA试剂盒定量测定模型组小鼠脑组织和脑脊液中Aβ1-42的含量明显高于正常组,与正常组相比有极显著性差异。中药组小鼠脑组织和脑脊液中Aβ1-42的含量较模型组明显下降,说明益智汤治疗可以降低模型组小鼠脑组织APP、Aβ1-40和Aβ1-42的表达。3.益智汤可减轻APP695转基因小鼠脑组织超微结构的损伤光镜观察正常组小鼠大脑皮层组织结构清楚,神经细胞形态结构正常。而模型组小鼠大脑皮层和海马CA1区组织结构紊乱,神经元胞体缩小,胶质细胞增生；微血管管壁有炎性细胞分布,血管壁不整齐。中药组细胞排列较模型组整齐,形态较正常。电镜观察正常组大脑皮层神经元细胞膜完整清晰,核浆比例大,核内染色质分布均匀；细胞质内细胞器丰富,线粒体、突触结构清晰；有髓神经纤维的髓鞘连续、完整、均匀。模型组小鼠大脑神经元细胞膜部分模糊不清,细胞核膜内褶,染色质边聚；细胞质内细胞器减少,各级溶酶体及脂褐素增加；线粒体肿胀、嵴断裂、空泡样变,部分突触变性,突触前后膜及间隙模糊不清；有髓纤维髓鞘破碎不连续；毛细血管不规则,呈现局部管壁增厚。中药组超微结构病变比模型组减轻。4.益智汤减少APP695转基因小鼠脑组织的细胞凋亡免疫组化结果显示模型组小鼠脑组织Caspase3阳性细胞数量明显多于正常对照组,着色深度也强于正常对照组；而益智汤治疗组Caspase3阳性细胞数量较模型组较少,着色强度也弱于模型组。Tune1法显示模型组小鼠皮层、海马部位有较多Tune1强阳性细胞,说明模型组小鼠脑组织出现较多凋亡细胞。与模型组比较,中药组Tune1强阳性细胞明显减少,显示中药益智汤治疗可减少模型组小鼠脑组织Caspase3的表达及神经细胞的凋亡。5.益智汤可以减少Aβ对体外培养神经细胞的毒性作用培养胎鼠大脑皮质神经元细胞贴壁良好,分布均匀,结构清晰,突触间联系密切。加入Aβ25-35(5、10、20及40μmol/L)24h后,细胞生长状态不良,细胞肿胀变性,MTT检测细胞活性下降、LDH漏出率增加,随着Aβ25-35浓度的增加,细胞活性明显降低,LDH漏出率也明显增加,显示了Aβ25-35的毒性作用。选用10μmol/LAβ25-35复制AD细胞模型,加入益智汤治疗大鼠血清后,可以明显提高模型组细胞活力、降低LDH漏出率,改善细胞形态学异常。结论APP695转基因小鼠出现学习记忆能力减退,脑组织APP及Aβ表达增高,出现超微结构的病理改变及神经细胞凋亡,是较为理想的AD模型动物。益智汤可以降低模型组小鼠脑组织APP及Aβ的表达水平,改善其超微结构病理变化及抑制脑组织神经细胞凋亡,这是其改善APP695转基因小鼠学习记忆能力的基础。Aβ在脑微血管壁沉积,损伤血脑屏障,加重了Aβ的毒性作用,益智汤可以减轻血脑屏障的损伤。同时益智汤大鼠血清可以直接抑制Aβ的细胞毒性,发挥神经保护作用。以上结果说明益智汤通过多靶点减轻Aβ的毒性作用发挥神经保护功能,中药补肾填髓、化浊开窍法治疗AD具有实验依据。更多还原

【Abstract】 Alzheimer disease(AD) is a kind of degenerative disease of central nervous system, the major pathological changes in AD are the formation of senile plaques(SP), neurofibrillary tangles(NFT) and loss of synapase and neurons in the cerebral cortex and subcortical structure, Which can severely impair the cognitive function and cause psychical and behavioral abnormality of the patient. The plaques are composed primarily of a small protein called amyloid-beta (Aβ). Aβhas toxicities to neurons and play important role in neurodegeneration and associated with cognitive and behavioral abnormalities. The evidence shows that intraparenchymal deposition of Aβis the major pathological hallmarks and plays a crucial part of the pathogenesis in AD. Traditional Chinese Medicine (TCM) considers that AD occurs in the brain and is closely connected with the deficiency of kidney and excess in superficiality in terms of its pathogenesis. Reinforcing the kidney to replenish marrow, removing blood and phlegm stasis to enrich the brain are the regular therapies. YiZhi fang is mainly composed of Herba Epimedii(Yinyanghuo), Panax quinquefolium(Xiyangshen), Barbary Wolfberry Fruit(GouQi), Rhizoma(Chuanxiong), Tall Gastrodia Tuber(Tianma) and Acorus tatarinowii Schott(Shichangpu), Which has the function of invigorating kidney to replenish marrow and removing phlegm and blood stasis to enrich the brain. The experiment focuses on the curative mechanism of YiZhi decoction to APP695 transgenic mice and provides a scientific ground and theoretical basis in clinical applications.Materials and Methods:1. Experimental study of the protective effect and its mechanism of YiZhi decoction on APP695 transgenic mice:APP695 transgenic mice (13 months old) were randomly divided into model group and YiZhidecoction group, and negative transgenic mice (C57BL/6J) were included into normal group. The mice in YiZhi decoction group were given intragastrically YiZhi decoction (0.1g/kg) for 3 months, and the model group and normal group were given equal quantity of distilled water. (1)The learning and memory behavior of mice were examined by using Y-type electric maze test and LashleyⅢwater maze test. (2) Applying immunohistochemical technique, the expressions of APP, Aβ1-40, Aβ1-42 and Caspase3 in mouse cerebral cortex and hippocampal CA1 were observed by light microscope, using Image analysis their integral optical density in all group. (3)The changes of cell morphology were detected by HE stain and were observed by light microscope, the ultrastructure in cerebral cortex of the mice was observed by the transmission electronic microscope. (4)The levels of Aβ1-42 in cerebrospinal fluid(CSF) by using ELISA Assay Kit. (5)Cerebral neurocyte apoptosis in all group were detected with Terminal deoxynucleotidel transferase mediated uridine nucleotide end labeling(TUNEL).2. Experimental study of the YiZhi decoction rat serum on primary cultured neural cells induced by Aβ25-35:The primary cultured fetal rat cortical neurons were growth for 8 days. The neurons were treated with aggregated Aβ25-35 with different concentration for 24h. The morphologic changes of fetal rat cortical neurons induced by Aβ25-35 were observed by light microscope. Using MTT assay detected the changes of vitality of cells and detected the leakage of LDH. And By using the same means, YiZhi decoction rat serum as a protective effect, we also detected the survival rate and the leakage of LDH in all groups of primary cultured fetal rat cortical neuron induced by Aβ25-35 and YiZhi decoction rat serum, to explore the protective effect of YiZhi decoction rat serum on damage induced by Aβ25-35.Objectives:1. YiZhi decoction can improve learning and memory capacity of the model mice:The results of Y-type electric maze test showed that the number used to indicate learning ability increased and the number used to indicate memory ability decreased in model group as compared with normal group (P<0.01), while the number used to indicate learning ability decreased and the number used to indicate memory ability increased in YiZhi decoction group as compared with model group. The results of Lashley III water maze test showed that the numbers of entering to blind-ending and lengthened latency in model group were more and longer than those in normal group, but those in YiZhi decoction group were considerably fewer and shorter than those in model group. The two means indicate that APP695 transgenic mice has learning and memory deficit and coincide with AD clinical feature, YiZhi decoction can improve learning and memory capacity of the model mice.2. YiZhi decoction can down-regulate the expressions of APP, Aβ1-40 and Aβ1-42 in APP695 transgenic mice:The number of APP, Aβ1-40 and Aβ1-42 positive cell and the degree of staining by their antibody in the cortex and hippocampal CA1 in the model group were higher than those in the normal group, using Image analysis their integral optical density (IOD). The levels of APP, Aβ1-40 and Aβ1-42 expression in the cortex and hippocampal CA1 of the model group obviously increased as compared with the normal group(P<0.01). the expression level of APP, Aβ1-40 and Aβ1-42 in YiZhi dectaion group were significantly lower than those in the model group(P<0.05). The levels of Aβ1-42 in cerebrospinal fluid(CSF) of model group were significantly higher than that in the normal group and YiZhi decoction group by using ELISA assay kit((P<0.05). The results indicate YiZhi decoction can down-regulate the level of APP, Aβ1-40 and Aβ1-42 in APP695 transgenic mice.3. YiZhi decotaion can relieve brain tissue morphological changes in APP695 transgenic mice:By the light microscope the modality configuration of cells in the Cerebral cortex and hippocampal CA1 in the normal group were normal, the neurons had regular arrangement with clear edges and obvious nucleus. But in model group the neurons had irregular arrangement and the edge of many cells was unclear, and there were infiltration of inflammatory cells in blood vessel walls, some neurons were atrophied; and those changes in the YiZhi decoction group were better than those in the model group, YiZhi decoction could improve brain tissue morphological changes in APP695 transgenic mice.By the electron microscope in normal group mice the cell in cerebral cortex were normal, the nucleus was round and big, the normal and abnormal chromatin was obvious, the configurations of endoplasmic reticulum, mitochondrion and synapses were also normal, both presynaptic and postsynaptic membranes were clear. In model group, the cells were atrophied and dwindled with obvious borderline, the swollen, disrupted mitochondria, active microglia, degenerative synaptic structure and disarranged microtubule were frequently found, the presynaptic and postsynaptic membranes were illegible; cell organelles reduced but the deposition of lipofuscin granules increased, the basement membranes of capillaries was irregular and had some deposits; Myelin sheaths were loose, part of myelin fragments completely separated, microtubule and microfilaments showed particle dissolution. These pathological changes in the brain of YiZhi decoction treat group mice were not so apparent and were similar to model group.4. YiZhi decoction can decrease cerebral neurocyte apoptosis in APP695 transgenic mice to protect cerebral neurocyte:The number of Caspase3 positive cell and the degree of staining by Caspase3 antibody increased in the brain of model group as compared with the normal group(P<0.01). The expression level of Caspase3 in YiZhi decotaion group was significantly lower than that in the model group (P<0.05). The TUNEL positive cells in model group increased than those in normal group(P<0.01). The TUNEL positive cell decreased in YiZhi dectoaion group than that in model group. The results indicate YiZhi deoctaion can decrease cerebral neurocyte apoptosis in APP695 transgenic mice to protect cerebral neurocyte.5 YiZhi decoction rat serum can protect the primary cultured neural cells from the damage induced by Aβ25-35:The primary cultured fetal rat cortical neuron were performed with Aβ25-35 (5,10,20, 40μmol/L) for 24 hours, the growth state is bad. The activity of cultured neurons were decreased and LDH leakage increased in culture solution, and the cell viability decreased gradually when the concentration of Aβ25-35 raised up, there was significantly difference between model group and normal control group(P＜0.01). using 10μmol/L Aβ25-35 to made AD model cells, added YiZhi decoction rat serum, The activity of cultured neurons were higher in YiZhi decoction group than that in Aβ25-35 group, the growth state is good. The activity of nerve cells increased and LDH activity decreased by comparison with the experimental control group, which indicate that the YiZhi decoction rat serum could protect the cells from the damage induced by Aβ25-35 through abatting Aβnerotoxicity.Conclusion: APP695 transgenic mice has learning and memory deficit, increase the level of APP and Aβexpression, and has cerebral pathological changes and neurocyte apoptosis in brain, so it is a good animal model for AD. YiZhi decoction can regulate the level of APP and Aβ, can relieve cerebral pathological changes and decrease cerebralneurocyte apoptosis to protect cerebral neurocyte. That is the basement to improve learning and memory ability. YiZhi decoction rat serum could protect the cells from the damage induced by Aβ25-35. So YiZhi decoction can antagonize toxicity of Aβon neuron and protect nerve cell. The study provides experimental support for reinforcing the kidney to replenish marrow, removing blood and phlegm stasis to provide a scientific ground for clinical treatment of AD.更多还原