通过两种痴呆症模型比较六味地黄颗粒和通络救脑滴丸的疗效特点

【作者】 狄波；

【导师】 李澎涛；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 1.背景老年痴呆症包括：轻度认知障碍、阿尔茨海默病(Alzheimer’s disease,AD)、血管性痴呆,以及由其他疾病,如帕金森病等导致的痴呆,其中以AD患者最为多见。该病是以认知功能缺损为主要症状的疾病,常伴有精神、行为和人格异常,严重影响日常生活或工作能力。全球患病人数庞大,造成了巨大的社会、经济、家庭压力。现代医学研究表明,AD发病主要与β-淀粉样蛋白(amyloid-β, Aβ)在脑组织异常沉积、脑内神经血管单元(neurovascular unit,NVU)功能异常导致Aβ清除障碍、胆碱能神经元受损、氧化应激、神经炎症反应或基因突变等因素有关,为此研究者针对调节Aβ代谢、改善胆碱能神经元功能或抗氧化应激等多方面研制了多种药物,不过目前对该病的治疗尚无切实有效的措施。中医从整体出发,认为老年痴呆症的病位在脑,发病与肾、心、脾、肝等脏腑的功能失调有密切关系。其病因以内因为主,或因肾精亏耗、气血不足、痰瘀交阻,脑髓失养；或因七情内伤、久病耗损、年迈体虚,脑髓失充,而致气、血、痰、瘀等病邪为患。并由此制定了补肾填精,解郁散结的总体治法,临床发现多种中药复方对该病显示了独特的疗效。本研究在老年痴呆症系“肾精不足”或“毒损脑络”而发病的中医病机指导下,结合两种拟老年痴呆症小鼠模型——肾虚精乏脑髓失充证(SAMP8小鼠)模型和毒损脑络证(双海马注射Aβ1-40的SAMR1小鼠)模型,探讨两种治疗老年痴呆症常用的不同功效中药复方——具有补肾填精功效的六味地黄颗粒(LWDH)和解毒通络功效的通络救脑滴丸(TLJN),对不同拟老年痴呆症模型的作用特点及可能的药效靶点,并比较这两种相同品系小鼠,在不同造模因素诱导下的神经行为学差异,寻找不同模型小鼠对造模因素反映的特征,以期为中药复方药效评价指标体系提供证据。2.目的1)确认增龄对两种拟老年痴呆症小鼠的影响；2)比较两种拟老年痴呆症模型小鼠的神经行为学及发病相关病理环节的差异；3)比较两种不同功效中药复方的作用特点,并根据动物模型的相关病理环节,寻找中药复方可能的药效靶点。3.方法1)使用行为学Morris水迷宫和跳台实验,比较2月龄、6月龄及8月龄Aβ注射模型和SAMP8模型与同月龄正常对照组在Morris水迷宫实验逃避潜伏期、寻台成功率、游速和跳台潜伏期、错误次数、错误区时间等指标的差异。2)对8月龄Aβ注射模型和SAMP8模型,均设立对症给药和非对症给药组,即两种模型分别均给予LWDH和TLJN治疗,使用行为学Morris水迷宫和避暗实验,比较上述各组在Morris水迷宫实验逃避潜伏期、寻台成功率、游速和避暗潜伏期、错误次数、错误区时间等指标的差异。3)使用免疫组织化学方法,标记上述各组小鼠脑组织Aβ、APP、BACE1、IDE、LRP1、RAGE、apoE、α2M等Aβ代谢相关蛋白的表达情况,并比较不同功效中药复方治疗组的差异。4)使用蛋白免疫印迹方法,分别检测上述各组小鼠海马和皮层APP、BACE1、IDE、LRP1、RAGE、apoE、α2M等蛋白的表达情况,并比较不同功效中药复方治疗组的差异。4.结果1)与对照组比较,8月龄Aβ注射模型和SAMP8模型在水迷宫逃避潜伏期、寻台成功率,跳台实验潜伏期、错误区时间及避暗实验潜伏期、错误次数、错误区时间等行为学检测指标,脑组织Aβ、APP、BACE1、IDE、LRP1、RAGE、apoE和a2M等蛋白表达上,均表现出显著性差异。在跳台实验和避暗实验错误区时间这个指标上,8月龄Aβ注射模型与同月龄对照组相比有差异。在水迷宫游速这个指标上,8月龄SAMP8模型与同月龄对照组相比有差异。2)两种模型之间,与8月龄Aβ注射模型比较,同月龄SAMP8模型在水迷宫游速,跳台潜伏期,避暗错误次数、错误区时间和脑组织APP(皮层)、BACE1(皮层)、IDE(皮层和海马)、LRP1(海马)、RAGE(皮层和海马)及apoE (皮层)蛋白表达上,有显著性差异。3)对于8月龄Aβ注射模型而言,对症药物,即TLJN,在调节模型小鼠避暗潜伏期、错误次数、错误区时间等行为学检测指标,脑组织APP、BACE1、IDE、LRP1、RAGE和apoE等蛋白表达上,发挥了显著作用。对于8月龄SAMP8模型而言,对症药物,即LWDH,在调节模型小鼠水迷宫逃避潜伏期、寻台成功率和跳台实验潜伏期等行为学检测指标,脑组织Aβ、APP、BACE1、IDE、LRP1、RAGE和α2M等指标上,发挥了显著作用。4)两种不同功效中药复方治疗组之间,对于8月龄Aβ注射模型而言,对症药物TLJN在改善该模型小鼠避暗潜伏期及脑组织皮层和海马APP表达、皮层apoE表达、海马BACE1、IDE和RAGE表达水平上,发挥了显著作用,而非对症药物LWDH对上述指标的调节作用不如TLJN;对于8月龄SAMP8模型而言,对症药物LWDH在改善小鼠水迷宫寻台成功率、避暗潜伏期及脑组织皮层和海马APP、BACE1、IDE表达、海马Aβ表达水平上,发挥了显著作用,而非对症药物TLJN对上述指标的调节作用不如LWDH。5.结论1)增龄对SAMP8小鼠和双海马注射Aβ1-40的SAMR1小鼠行为学改变有显著影响。2)不同证型的模型之间,8月龄Aβ注射模型和SAMP8模型,均表现出了空间学习记忆、对电刺激的记忆、被动逃避反应能力缺失,及Aβ代谢相关蛋白的表达异常。但是,与Aβ注射模型比较,SAMP8模型的活动度相关指标(如,水迷宫游速、避暗错误次数等)下降；增加脑内Aβ水平的相关蛋白(如APP、BACE1、RAGE等)表达更高；降低脑内Aβ水平的相关蛋白(如IDE、LRP1、apoE等)表达更低,反应了SAMP8模型Aβ代谢障碍更严重,及肾虚精乏证的行为学特征。3)不同功效中药复方之间,对于8月龄Aβ注射模型和SAMP8模型,对症药物均发挥了纠正Aβ代谢障碍的作用,表现在降低Aβ生成相关蛋白APP和BACE1的表达；增加Aβ降解相关蛋白IDE的表达等。非对症药物对上述指标未发挥显著改善作用,甚至还有反作用。提示,脑组织APP、BACE1、IDE等与Aβ生成及降解相关的蛋白,可作为评价不同功效中药复方的特异性指标,反应药物的药效特点。4)对于脑内清除Aβ的重要途径LRP1系统而言,在调节多数指标上,不同功效中药复方分别对两种模型,均发挥了作用,且作用趋势一致。仅在8月龄Aβ注射模型的皮层apoE、海马RAGE表达上,及SAMP8模型的皮层LRP1表达上,表现出了对症药物的优势。提示,可能因为LRP1和RAGE及apoE、α2M等蛋白之间有密切的相互作用,发生病理改变时,相互影响也更复杂,导致不同功效中药复方在不同模型上未显示出特异性疗效。但该系统指标可作为评价药物整体疗效的参考指标。5)对于行为学相关指标而言,不同功效中药复方对两种证型的模型,在8月龄Aβ注射模型的避暗潜伏期、错误次数、错误区时间等指标,及SAMP8模型的水迷宫逃避潜伏期、寻台成功率和避暗潜伏期、错误次数等指标上,表现出了对症药物的优势。提示对症药物对于改善相应证候模型小鼠的空间学习记忆和被动逃避反应能力上有作用,有助于评价不同功效中药复方的药效特点。总之,这两种拟老年痴呆症小鼠模型在Aβ代谢相关蛋白的表达和行为学上有各自的病理特点,不同功效中药复方对不同模型的起效靶点也有特异性,且对症给药对于改善模型行为学和Aβ生成及降解的环节上,发挥特异性作用,因此这些指标可作为评价不同功效中药复方药效的特异性指标。而LRP1系统的各指标,在两种模型上,未反映出不同功效中药复方的药效特点,作为评价指标,特异性欠佳。所以,对于中药复方药效评价指标而言,应根据药效特点和模型证候进行选择,以期更客观地评价中药复方药效,更准确地发现药物作用靶点和特点。更多还原

【Abstract】 1. BackgroungDementia is a complex disease which be characterized by impairments in cognition and behavior. Behavioural and psychological symptoms of dementia are present in the course of the illness in most patients with dementia. The incidence of dementia among the elderly population is rising rapidly around the world. Due to the numerous cases of patients, there are more and more burden of society, economy and family. Alzheimer’s disease(AD) is the leading type of dementia. AD is a neurodegenerative disorder of the aged that has multiple factors, which contribute to AD’s etiology in terms of initiation and progression. There are several hypotheses have been formulated to explain this debilitating disease, and those hypotheses often have overlapping features. The best-known hypothesis to explain AD is the Amyloid Cascade Hypothesis which involves the role of the accumulation of amyloid-βpeptide(Aβ) in the brain. Other theories include Dysfunction of Neurovascular Unit which suggests manifold neurovascular pathogenic cascades for AD, cholinergic hypothesis, the role of oxidative stress or neuroinflammation, genes mutation and so on. Current therapeutics on the market, including cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists and productions of modulating Aβmetabolism, provide symptomatic relief but do not alter progression of this disease. Therefore, progress in the areas of prevention and disease modification may be important. Based on Traditongal Chinese medicine, dysfunction of brain, kidney, heart, spleen and liver all may due to AD.Deficiency of kidney essence, asthenia of qi and blood, sputum and blood stasis, internal injury due to seven emotions, prolonged illness consuming healthy qi all would impair the brain. The basic therapeutic methods is Invigorating kidney and supplementing essence, relieve accumulation and resolve stagnation. Traditongal Chinese medical prescriptions are multifunctioning that could theoretically intervene at any of a number of sites to abate the changes associated with the development of AD and have a better effection.This experiment is guided by the pathogenesis theories of Consuming of Kidney Essence and Toxin Hurts Brain Collaterals to study SAMP8(model-P) & hippocampus injection of Aβ1-40 in SAMR1(model-A) models, to compare the actions of Liu Wei Dihuang granule(LWDH) and Tong Luo Jiunao dripping pill(TLJN), and to find the feature of those prescriptions. 2. Objective1) To test the correlation of aging and cognition impairments in two models;2) To compare the effect of behavioral test and morphology of two models;3) To compare the speacil function of two prescriptions, and ascertain the potential drug targets.3. Methods1) To investigate the diversity of 2-month-old,6 month-old and 8 month-old SAMP8 and SAMR1 mice using Morris water maze and passive avoidance test(Step-up test).2) To investigate the discrepancy of 8 month-old model-P and model-A treated by LWDH(PL group & AL group) and TLJN(PT group & AT group) using Morris water maze and passive avoidance test(Step-through test).3) To compare the difference in all group on expression of Aβ,APP, BACE1, IDE, LRP1, RAGE, apoE and a2M using immunohistochemistry.4) To compare the difference in all group on level of APP, BACE1, IDE, LRP1, RAGE, apoE and a2M using Weatern blotting.4. Results1) To compare with the N group, there are diversity in MWM escape latency, success rate and Step-up test latency, wrong frequency, and Step-though test latency, wrong frequency of 8-month-old model-P and model-A. To compare with the N group, there are diversity in both Step-up test and Step-though test wrong time of model-A. To compare with the N group, there are diversity in MWM swimming speed of model-P.2) To compare with the A group, there are difference between model-A and model-P on MWM swimming speed, Step-up test wrong frequency, Step-though test wrong frequency, wrong time and expression of APP(cortex), BACE1(cortex), IDE(cortex and hippocampal), LRP1(hippocampal),RAGE(cortex and hippocampal) and apoE(cortex).3) The irregular expression of Aβ, APP, BACE1, IDE, LRP1, RAGE, apoE and a2M in brains of model-P and model-A. The regulated expression of APP, BACE1, IDE, LRP1 RAGE and apoE were shown in AT group as well as the regulated expression of Aβ, APP, BACE1, IDE, LRP1, RAGE, and a2M were tested in PL group. TLJN have function on model-A by regulated Step-though test latency and expression of APP, BACE1, IDE, LRP1,RAGE and apoE. LWDH have function on model-P by regulated MWM success rate, Step-though test latency and expression of Aβ,APP, BACE1, IDE and RAGE.5. Conclusions1) There are correlation of aging and cognition impairments in two models;2) Model-P and model-A have severely impaired acquisition and retention of spatial learning task and passive avoidance response, and neurochemical. So SAMP8 maybe a more severely model.3) These two traditongal Chinese medical prescriptions have significant effection, due to selecting of treatment based on the differential diagnosis. The expreesion of APP、BACE1、IDE coule be useful develop and evaluate potential new formulations.4) There are few difference between LWDH and TLJN in clearance of Aβby LRP1 system. So the evaluation for traditongal Chinese medical prescriptions should be unambiguous.5) The behavioral test maybe unsuitable as a evaluation for different traditongal Chinese medical prescriptionsIn conclusion, SAMP8 and hippocampus injection of Aβ1-40 in SAMR1 mice have difference on MWM swimming speed, Step-up test wrong frequency, Step-though test wrong frequency, wrong time and expression of APP, BACE1, IDE, LRP1,RAGE and apoE. To hippocampus injection of Aβ1-40 in SAMR1 mice, TLJN have function by regulated Step-though test latency, and expression of APP, BACE1, IDE, LRP1,RAGE and apoE. On the other hand, to SAMP8 mice LWDH have function by regulated MWM success rate, Step-though test latency and expression of Aβ, APP, BACE1, IDE and RAGE. These two models and two traditongal Chinese medical prescriptions have their own characteristics.更多还原