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

发布时间：2018-04-18 03:38

本文选题：老年痴呆症 + 快速老化小鼠（SAM）　；参考：《北京中医药大学》2012年博士论文

【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。
[Abstract]:1 . Background
Alzheimer ' s disease ( AD ) , vascular dementia , and dementia caused by other diseases , such as Parkinson ' s disease , have been studied .
or can be caused by seven - case internal injury , long - term disease consumption , aging body deficiency , brain marrow loss , qi , blood , phlegm , blood stasis , etc . , and the general treatment method for tonifying kidney and replenishing essence and resolving stagnation is developed , and various traditional Chinese medicine compounds are found to have unique curative effect on the disease .
The purpose of this study was to explore the effects of two kinds of traditional Chinese medicine compound _ ( LWDH ) and toxin - impaired brain collateral syndrome ( SAMR1 mouse ) model of two kinds of senile dementia mice , and to explore the effects of two kinds of traditional Chinese medicines for the treatment of senile dementia .
2 . Purpose
1 ) Confirm the effect of aging on two kinds of senile dementia mice ;

2 ) comparing the differences of neurobehavioral and pathological links between the two models of Alzheimer ' s disease ;

and 3 ) comparing the functional characteristics of the traditional Chinese medicine compound with two different efficacies , and looking for the possible drug effect target point of the traditional Chinese medicine according to the relevant pathological links of the animal model .
3 . Methodology
1 ) Using the behavioral Morris water maze and the skip experiment , the differences of the latency , the success rate , the latency , the number of errors and the time of the wrong area were compared between the two - month - old , 6 - month - old and 8 - month - old A尾 injection models and the normal control group of the same month in Morris water maze test .
2 ) In the 8 - month - old A - 尾 injection model , the two models were treated with both LWDH and TLJN , the behavioral learning Morris water maze and the avoiding - dark experiment were used to compare the differences between the above groups in Morris water maze test to avoid the latency , the success rate , the swimming speed and the latency of avoiding dark latency , the number of errors , and the time of the wrong area .
3 ) Using immunohistochemical method , the expression of A尾, APP , BACE1 , IDE , LRP1 , RAGE , apoE , 伪2M , and so on in brain tissue of each group were marked .
4 ) Using Western blot method , the expression of APP , BACE1 , IDE , LRP1 , RAGE , apoE , 伪2M in the hippocampus and cortex of the above groups were detected , and the difference of different efficacy Chinese herbal compound treatment groups was compared .
4 . Results
1 ) Compared with the control group , the 8 - month - old A尾 injection model and the 8 - month - old A - 尾 injection model showed significant difference compared with the control group . The 8 - month - old A - 尾 injection model was different from the control group in the same month .
2 ) There was a significant difference between the two models , which was compared with the 8 - month - old A尾injection model , and the same month - old model was significantly different in the water maze tour speed , the latency of the platform , the number of avoiding dark errors , the time of the wrong area and the brain tissue APP ( cortex ) , the BACE1 ( cortex ) , the IDE ( cortex and hippocampus ) , the LRP1 ( hippocampus ) , the RAGE ( cortex and hippocampus ) and apoE ( cortex ) protein expression .
3 ) For the 8 - month - old A尾 injection model , the therapeutic drugs , TLJN , play a prominent role in regulating the expression of protein such as the latent period , the number of errors , the time of the wrong area and so on in the model mice . For the 8 - month - old , the symptomatic drug , i.e . , LWDH , plays a prominent role in the indexes such as the escape latency of the water maze in the model mice , the success rate of the search and the latency of the platform experiment , and the indexes such as A尾, APP , BACE1 , IDE , LRP1 , RAGE and 伪2M in brain tissue .
4 ) For the 8 - month - old A 尾 injection model , TLJN plays a prominent role in improving the expression level of the mouse ' s dark latency and brain cortex and hippocampus , apoE expression , the expression level of BACE1 , IDE and RAGE in the hippocampus , and plays a prominent role in improving the success rate of the mouse water maze , avoiding dark latency and the level of the brain cortex and hippocampus APP , BACE1 , IDE and hippocampus A尾expression , and the non - symptomatic drug TLJN is not as good as the LWDH .
5 . Conclusions
1 ) There was a significant effect on the behavioral changes of SAMR1 mice injected with A尾 1 - 40 by age - increasing mice and hippocampus .
2 ) Between models of different types , 8 - month - old A.beta . injection model and 8 - month - old model showed spatial learning and memory , loss of passive avoidance response ability and abnormal expression of A尾metabolism - related protein . However , compared with A尾injection model , the activity degree - related indexes ( e.g . , water maze tour speed , avoiding dark error number , etc . ) were decreased compared with A尾injection model .
Increased expression of related proteins ( e.g . , APP , BACE1 , RAGE , etc . ) in brain A尾levels was increased ;
The lower expression of related protein ( such as IDE , LRP1 , apoE , etc . ) in brain A 尾 level was lower , and the behavioral characteristics of A尾metabolism disorder and kidney deficiency syndrome were studied .
3 ) For the 8 - month - old A - 尾 injection model and the 8 - month - old A - 尾 injection model , the action of correcting the metabolic disorder of A尾was exerted on the symptomatic drugs , which showed that the expression of APP and BACE1 associated with A尾was decreased .
It is suggested that the protein of APP , BACE1 , IDE and other proteins related to the formation and degradation of A尾can be used as a specific index for evaluating the compound of different efficacies of Chinese medicine and the drug effect of the reaction medicine .
4 ) For the LRP1 system , which is an important pathway for the elimination of A尾in the brain , the effects of different traditional Chinese medicine combinations on the regulation of most indexes are consistent . Only in the cortical apoE , the hippocampus RAGE expression of the 8 - month - old A尾 injection model and the expression of the cortical LRP1 of the model of the 8 - month - old A尾 injection , the interaction between the two traditional Chinese medicine compounds is more complicated , which leads to the lack of specific therapeutic effect on different models .
5 ) For the indexes of behavior - related indexes , the models of the two types of traditional Chinese medicine compound are shown , and the advantages of the drugs are shown in the indexes such as the hidden latency , the number of errors , the time of the wrong area and the like of the 8 - month - old A.beta . injection model , the success rate of the search and the latency of avoiding dark latency and the number of errors , etc . The indications are helpful for improving the spatial learning and memory and the passive avoidance response capability of the corresponding syndrome model mice , and help to evaluate the efficacy characteristics of the Chinese traditional Chinese medicine compound .
In conclusion , the two models of Alzheimer ' s disease have their own pathological characteristics in the expression and behavior of A尾metabolism - related proteins , and the different effects of traditional Chinese medicine are specific for the improvement of model behavior and the generation and degradation of A尾. Therefore , these indexes can be used as the indexes for evaluating the efficacy of Chinese traditional Chinese medicine compound . Therefore , the indexes of the LRP1 system can be selected according to the drug effect characteristics and the model syndrome , so as to objectively evaluate the compound drug effect of the traditional Chinese medicine and more accurately find the target point and the characteristic of the drug action .

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R277.7;R-332

【参考文献】