固本健脑法对AD大鼠模型的作用及其机制研究

发布时间：2018-03-03 16:02

本文选题：阿尔茨海默病　切入点：固本健脑法　出处：《湖北中医药大学》2015年博士论文　论文类型：学位论文

【摘要】：目的随着社会人口的快速老龄化,阿尔茨海默病(Alzheimer's disease, AD)因其高患病率及对社会、家庭的影响日益受到广泛重视。因此,全世界均意识到加强研究AD的病因与发病机制,以及开发有效的防治措施和药物的重要意义。本研究深入探讨固本健脑法治疗AD的作用机理,阐释固本健脑治则治法理论的科学内涵,为寻求中医改善学习记忆、治疗AD的方药提供依据,丰富中医固本理论及中医防治AD理论的内容。方法1.理论研究：采用文献研究方法,系统回顾中医学从先后天之本认识老年性痴呆的理论,结合团队相关研究成果入手,系统阐述固本健脑法治则治法理论。2.实验研究：围绕tau蛋白过度磷酸化损伤是AD发病的核心机制,以抑制tau蛋白过度磷酸化、保护树突棘的信号传导功能为靶点,选用安理申作为西药对照,运用固本健脑法对AD大鼠模型进行干预,观察其对AD模型的作用并探讨其作用机制。取12月龄SPF级Wistar大鼠70只,雌雄各半。适应性喂养1w后,随机分为7组,分别为正常组、空白对照组、模型组、固本健脑液高剂量组(简称固高组)、固中组、固低组和安理申组,每组10只。正常组不予处理。模型组和各治疗组给予双侧海马脑立体定位注射5μg/μl浓度的A β1-42：复制AD模型,空白对照组注射等位点等剂量生理盐水。3d后,各治疗组分别按照10ml/kg·d给予相应药物浓度的固本健脑液和安理申混悬液灌胃,模型组与空白对照组则按照相同剂量给予生理盐水灌胃,连续4w。灌胃结束后,对各组大鼠分别进行水迷宫定位航行实验和空间探索实验,观察固本健脑法对AD大鼠模型行为学的影响。高尔基染色法观察各组大鼠海马神经元树突棘形态变化及密度改变。RT-PCR法检测各组大鼠海马EphB2、PARP-1水平变化。Western Blot法检测各组大鼠海马总tau、P-tau(Ser199, Ser396,Thr231位点)、EphB2、PARP-1表达变化。观察固本健脑法对AD大鼠模型学习记忆能力、树突棘形态及tau蛋白过度磷酸化的作用,以探讨其防治AD的作用机制。结果1.固本健脑法对AD大鼠模型行为学的影响定位航行实验中,模型组大鼠找到水下隐匿平台潜伏期明显长于正常组(P0.01),各治疗组潜伏期明显短于模型组(P0.01),且固高组、固中组潜伏期短于安理申组(P0.01)；目标象限游泳时间百分比比较,模型组明显低于正常组(P0.01),各治疗组明显高于模型组(P0.01),且固中组高于安理申组(P0.05)。空间探索实验中,模型组大鼠找到原平台潜伏期明显长于正常组(P0.01),各治疗组潜伏期明显短于模型组(PO.01),且固高组、固中组潜伏期短于安理申组(P0.05)；模型组大鼠穿越原平台位置次数明显少于正常组(P0.01),各治疗组潜伏期明显多于模型组(P0.01),且固高组、固中组多于安理申组(P0.01)；目标象限游泳时间百分比比较,模型组明显低于正常组(P0.01),各治疗组明显高于模型组(P0.01),且固高组、固中组高于安理申组(P0.05)。表明本研究的AD模型是成功的,固本健脑法能明显改善AD模型大鼠的学习记忆功能。2.固本健脑法对AD大鼠模型海马神经元树突棘的影响比较各组大鼠的神经元形态学发现,模型组大鼠海马区神经元树突棘密度明显低于正常组(P0.01),各治疗组树突棘密度明显高于模型组(P0.01),且固高组、固中组均高于安理申组(P0.01)。表明固本健脑法能明显改善AD大鼠海马神经元树突棘的形态。3.固本健脑法对AD大鼠模型海马区P-tau的影响Western blot法检测各组大鼠海马tau、P-tau并比较P-tau水平,结果模型组大鼠较正常组明显升高(P0.01),各治疗组明显低于模型组(P0.01),且固高组、固中组均低于安理申组(P0.01)。4.固本健脑法对AD大鼠模型海马EphB2表达的影响Western blot与RT-PCR法检测均显示：模型组大鼠海马EphB2表达较正常组明显降低(P0.01),各治疗组大鼠海马EphB2表达明显高于模型组(P0.01),且固高组、固中组大鼠海马EphB2表达均高于安理申组(P0.05)。5.固本健脑法对AD大鼠模型海马PARP-1表达的影响Western blot与RT-PCR法结果均显示：模型组大鼠海马PARP-1表达较正常组明显升高(P0.01),各治疗组大鼠海马PARP-1表达明显低于模型组(P0.05),且固中组高于安理申组(P0.05)。结论1.AD的发病与脾肾关系密切,先后天之本脾肾亏虚、脑髓失养,兼有痰瘀是其基本病机,固本健脑法是治疗该病的有效治法。2.固本健脑法能明显改善AD模型大鼠的学习记忆能力；提高海马神经元树突棘密度；升高海马EphB2表达,降低PARP-1水平,抑制tau蛋白过度磷酸化。3.固本健脑法能有效防治AD,改善临床症状,其机制可能与升高海马EphB2、降低PARP-1从而抑制tau蛋白过度磷酸化,保护神经元树突棘、保护神经元功能有关。
[Abstract]:Objective with the rapid population aging society, Alzheimer's disease (Alzheimer's, disease, AD) because of its high prevalence and the society, the influence of the family has received more and more attention. Therefore, all over the world are aware of strengthening the etiology and pathogenesis of AD has important significance, effective prevention measures and drugs and the development. Study the physic treatment mechanism of AD, explains the physic treatment scientific connotation of treatment theory, to improve the learning and memory of TCM, provide the basis for prescription in the treatment of AD, the rich Chinese medicine theory and the theory of TCM Prevention and treatment of AD. 1. research methods: literature research method, system from the review of the theory of traditional Chinese medicine has a knowledge of dementia, with team related researches, systematically expounded the physic treatment theory.2. experimental study on the rule of law: focus on tau protein Hyperphosphorylation of damage is the core mechanism of AD, to inhibit tau phosphorylation and protect the signaling function of dendritic spines as the target, selection of Aricept as western medicine control, using the physic method intervention on AD rat model, observe the effect of the AD model and explore its mechanism. 70 rats, 12 month old SPF Wistar female. Feeding after 1W, were randomly divided into 7 groups, including normal group, blank control group, model group, the physic liquid in high dose group (referred to as Gugao group), solid solid group, low dose group and donepezil group, n = 10 only. The normal group were not treated. The model group and the treatment group was treated with bilateral hippocampal brain stereotaxic injection of 5 mu g/ Mu l concentration of A beta 1-42: duplicate AD model, blank control group was injected with saline sites after.3d in different treatment groups were given corresponding drugs according to the 10ml/kg D concentration of the physic Liquid and Aricept suspension gavage, model group and blank control group with the same dose of saline, continuous intragastric administration of 4w. after the end of each rat were water maze navigation experiment and space exploration experiment, the physic method in observation of AD rat model for learning effects were observed in rat hippocampal neurons and morphological changes of dendritic spine density changes detected by.RT-PCR EphB2 in the hippocampus of rats of Golgi staining, PARP-1 level changes of.Western Blot tau were detected in the hippocampus of rats, P-tau (Ser199, Ser396, Thr231, EphB2 loci), expression of PARP-1. The physic method was used to observe the learning and memory the ability of AD rat model, effect of dendritic spine morphology and hyperphosphorylation of tau protein and to investigate its mechanism of prevention and treatment of AD. The effect of 1. methods to model the physic behavior of AD rats. In the experiment, the model group The rat found the latency of underwater is significantly longer than the normal group (P0.01), the treatment group was significantly shorter than that in the model group (P0.01), and Gugao group, solid group was shorter than Aricept group (P0.01); compare the percentage of swimming time in the target quadrant, the model group was significantly lower than normal group (P0.01), the treatment group was significantly higher than the model group (P0.01), and the solid is higher than the Aricept group (P0.05). Space exploration experiment, the rats in the model group to find the original platform was significantly longer than the normal group (P0.01), the treatment group was significantly shorter than that in the model group (PO.01), and high solid solid group. The incubation period is shorter than the Aricept group (P0.05); model group rats crossing the original platform number less than the normal group (P0.01), the treatment group was significantly more than the model group (P0.01 group), and high solid, solid group than Aricept group (P0.01); compare the percentage of swimming time in the target quadrant. Die Type group was significantly lower than normal group (P0.01), the treatment group was significantly higher than that in the model group (P0.01), and solid solid is higher than the high group, the Aricept group (P0.05). This study shows that the AD model is successful, the physic method can obviously improve the physic method of learning and memory function of.2. AD model the effects of rat hippocampal neurons of AD rat model of dendritic spine morphology of neurons were compared in rats found that neurons in hippocampus of model group rats spine density were significantly lower than the normal group (P0.01), the treatment group of spine density was significantly higher than the model group (P0.01), and solid solid group were higher than the high group. The Aricept group (P0.01) showed that the effect of the physic method. The morphology of.3. the physic method can obviously improve the hippocampal neuronal dendritic spines in AD rat model of P-tau AD in hippocampus of rats with Western blot method to detect the rat hippocampal tau, P-tau and P-tau level, results in the model group Rats increased significantly compared with normal group (P0.01), the treatment group was significantly lower than that of model group (P0.01), and solid solid group were lower than the high group, Aricept group (P0.01.4.) to detect the physic method on the expression of EphB2 in hippocampus of AD rat model and the effect of Western blot RT-PCR method showed that: the expression model of EphB2 in the hippocampus of rats significantly decreased compared with normal group (P0.01), EphB2 in hippocampus of rats in each treatment group was significantly higher than that in the model group (P0.01), and high solid solid group, expression of EphB2 in hippocampus of rats were higher than that of the Aricept group (P0.05) and Western blot RT-PCR method.5. the physic method on the expression of hippocampus PARP-1 AD rat model showed that the expression of PARP-1 in hippocampus of model rats increased significantly compared with normal group (P0.01), PARP-1 in hippocampus of rats in each treatment group was significantly lower than the model group (P0.05), and the solid is higher than the Aricept group (P0.05). Conclusion the incidence of 1.AD close relationship with spleen and kidney has A deficiency of spleen and kidney medulla dystrophy, both phlegm and blood stasis is the basic pathogenesis, the physic method is the physic method in treating the disease effective treatment.2. can significantly improve the learning and memory ability of AD rats; improve the hippocampal neuron dendritic spines; increased the expression of EphB2 in hippocampus, decreased the level of PARP-1. Inhibition of tau hyperphosphorylation of the physic method.3. can effectively control AD, improve the clinical symptoms, the mechanism may be related to increased hippocampal EphB2, decreased PARP-1 and inhibited tau phosphorylation and protection of dendritic spines, protect neuron function.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5;R-332

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