【作者】 赵清山；

【导师】 唐农；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2014， 博士

【摘要】 血管性痴呆(Vascular dementia, VD)是指在缺血性、出血性及急慢性缺血缺氧性脑血管性疾病引起的脑组织损害基础上产生的以高级神经认知功能障碍为主的一组临床综合征。在我国,血管性痴呆居痴呆的首位,且发病率程逐年上升之势,给个人、家庭和社会带来巨大的负担。因此,VD的防治日益受到人们的关注。研究表明,长时程增强(LTP)和学习、记忆过程密切相关,神经递质及其信号转导通路在LTP形成过程中起着关键的作用,特别是cAMP-PKA-CREB信号通路作为其中的一个重要信号调节系统,在突触可塑性中发挥重要作用,能促进神经细胞的存活、再生和分化,在调节学习和记忆形成过程中具有重要的作用。本课题从共振理论的角度探讨血管性痴呆的病机,并结合一气周流理论从肺的生理功能出发提出从肺论治血管性痴呆的理论基础和有效方剂益肺宣肺降浊方。在建立血管性痴呆大鼠模型的基础上,分别运用Morris水迷宫实验、光镜、电镜、TUNEL法检测大鼠行为学和海马形态学变化,并运用ELISA法和Western blot技术检测cAMP-PKA-CREB信号转导通路相关蛋白cAMP含量、PKA和p-CREB在大鼠海马区表达的变化,观察经益肺宣肺降浊方干预后VD大鼠cAMP-PKA-CREB信号转导通路学习记忆相关蛋白的表达变化,探讨该方改善VD的内在机制,进一步丰富VD的研究思路。为此,本文从以下两个方面进行了研究。1.文献研究本部分首先就历代中医关于VD的认识、中医药治疗血管性痴呆的研究现状进行了论述。其次,从人体血液循环共振理论的角度探讨血管性痴呆的病机,认为血管性痴呆属于中医伤寒三阴病的范畴,其病因在于颈动脉或脑动脉管腔狭窄、硬化,弹性下降,振动频率变高,血液无法充分进入大脑,脑部供血不足,影响其正常功能的发挥,进而逐渐出现记忆减退甚至痴呆,并结合一气周流理论从肺的生理功能与血管性痴呆关系出发提出从肺论治血管性痴呆的理论基础,在此基础上提出了益肺宣肺降浊方的设计原则和药物组成,并列举一则验案分析其具体运用。2.实验研究目的：在建立血管性痴呆大鼠模型的基础上,观察经益肺宣肺降浊方干预后VD大鼠cAMP-PKA-CREB信号转导通路学习记忆相关蛋白的表达变化,探讨该方改善VD的内在机制。方法：(1)选用SPF级SD雄性大鼠,适应性喂养后通过Morris水迷宫检测筛选剔除不会游泳及智力低下的大鼠。随机分为空白对照组和造模组,对造模组大鼠采用双侧颈总动脉夹闭再灌注方法制备血管性痴呆模型。造模后7d,存活的大鼠均再次进行Morris水迷宫测试,有显著学习记忆障碍的大鼠为血管性痴呆大鼠模型,将造模成功的大鼠随机分为模型组、中药高剂量组、中药中剂量组、中药剂低量组、西药对照组石杉碱甲组和脑复康组,连续灌药30天,进行Morris水迷宫行为学检测后取材。(2) Morris水迷宫检测：运用Morris水迷宫实验检测VD大鼠行为学变化,验证造模是否成功及益肺宣肺降浊对VD大鼠行为学的影响。(3)病理形态学结构的观察：分别运用光镜、电镜、TUNEL法观测大鼠海马形态学变化,并进一步判断VD大鼠是否存在形态学异常及益肺宣肺降浊方对其影响。(4) cAMP-PKA-CREB通路相关蛋白的检测：运用ELISA法检测各组大鼠海马cAMP含量的变化,运用Western blot技术检测各组大鼠海马PKA和p-CREB蛋白表达的变化,进一步判断VD大鼠是否存在cAMP-PKA-CREB信号转导通路的异常及益肺宣肺降浊方对该通路的影响。结果：(1) Morris水迷宫实验结果显示：与模型组对比,益肺宣肺降浊方组大鼠逃避潜伏期显著缩短,在撤除平台后穿越平台的次数显著增加；尤以中药高剂量组效果最为明显。(2)光镜、电镜、TUNEL法检测结果均显示：与模型组对比,益肺宣肺降浊方组大鼠海马神经细胞凋亡明显减少,尤以中药高剂量组效果最为明显。(3) ELISA法和Western blot法检测结果显示：与模型组对比,使用益肺宣肺降浊方干预后,cAMP含量、PKA和p-CREB的表达均有显著升高,尤以中药高剂量组效果最为明显。结论：(1) Morris水迷宫实验、光镜、电镜观察、TUNEL法、ELISA法和Western blot法等多种实验结果提示：益肺宣肺降浊方能显著提高血管性痴呆大鼠的学习记忆能力；能显著抑制大鼠海马神经细胞的凋亡,保护神经元；能显著提高cAMP-PKA-CREB信号转导通路学习记忆相关蛋白的表达。在此基础上,推测其可能机制为：益肺宣肺降浊方通过提高海马组织cAMP含量、PKA和p-CREB的表达,激活cAMP-PKA-CREB信号转导通路,促进LTP形成,保护神经元,从而改善VD大鼠的学习记忆能力。(2)益肺宣肺降浊方对血管性痴呆大鼠具有较好的治疗作用,提示血管性痴呆从肺论治是具有可行性,值得进一步发掘。更多还原

【Abstract】 Vascular dementia(VD),resulting from brain loss caused by varieties of cerebrovascular diseases, is an acquired intelligent damage syndrome and cognitive function defect.Cerebrovascular diseases lead to atherosclerosis, and ischemia or ischemia-reperfusion injury will appear, which results in dementia.Studies have shown that as one of the important signal pathways in the formation long-term potentiation (LTP) process,cAMP-PKA-CREB signaling pathway plays an important role in regulating the process of learning and memory formation,which can promote the survival of nerve cells, regeneration and differentiation.On the basis of blood circulation resonance theory and Qi cycle theory,this text presents the theory Treating VascularDementia from the physiological function of the lung and the and effective prescription Yifei Xuanfei Jiangzhuo fang.The aim of the present study was to investigate the effect of cAMP-PKA-CREB signaling pathway on learning and memory activities in a rat model of vascular dementia (VD) with a variety of experimental methods.First Part:Theory researchFirstly,recall ancient physicians knowledge about VD and discuss the research status of Chinese medicine treatment of vascular dementia.Secondly,we think that the pathogenesis of vascular dementia is that the carotid artery or stenosis of cerebral arteries,hardening,decreased flexibility,which causes higher vibration frequency,makes the blood can not fully enter the brain,lack of blood supply to the brain,affects the brain’s normal function,and then causes the memory loss even dementia.On the basis of Blood circulation resonance theory and Qi cycletheory,This text presents the theory Treating Vascular Dementia from the physiological function of the lung and the and effective prescription Yifei Xuanfei Jiangzhuo fang.Second Part:Empirical researchPurpose:Establish the vascular dementia rat model and observe the expression changes of protein associated with cAMP-PKA-CREB signaling pathway after the intervention of Yifei Xuanfei Jiangzhuo fang in VD rats.Method:(1) Selecting the big mice of Wistar,sets up the VD model,all the VD rats were randomly divided into six groups:Model group,the high doses group,the Moderate doses group,the lower doses group,the amount of pharmaceutical group,comparison group huperzine,comparison group pisacetam.All the rate were tested by Morris water maze after30days continuous perfusion;(2) Morris water maze test:Test the behavioral changes in VD rats by Morris water mazeverify the success and impact modeling Yifei Xuanfei Jiangzhuo VD rats learned behavior and observe the effect of Yifei Xuanfei Jiangzhuo on the behavior of VD rats,(3) Pathological structures:Observe the morphological changes in rat hippocampus and the effect of Yifei Xuanfei Jiangzhuo fang on the behavior of VD rats using the Light microscopy,electron microscopy,TUNEL method.(4) Detection of the proteins related to the cAMP-PKA-CREB pathway:Observe the expression changes of the protein cAMP,PKA,p-CREB,associated with cAMP-PKA-CREB signaling pathway after the intervention of Yifei Xuanfei Jiangzhuo fang in VD rats.Result:(1)Morris water maze test results:Compared with the model group,the escape latency of Yifei Xuanfei Jiangzhuo group were significantly shortened and the number across platforms after the removal of the platform significantly increas-ed;(2)pathological structures results:Compared with the model group,the number of hippocampal neuronal apoptosis of Yifei Xuanfei Jiangzhuo fang group were significantly reduced; (3)ELISA and Western blot results:Compared with the model group,the expression of the protein cAMP, PKA and p-CREB of Yifei Xuanfei Jiangzhuo fang group is significantly increased.Conclusion:(1) Results of all the tests show that:Yifei Xuanfei Jiangzhuo could Improve memory of vascular dementia rats significantly,inhibit apoptosis in rat hippocampal neurons,protect neurons;increase the expression of the proteins related to cAMP-PKA-CREB signaling pathway. Based on the research above,we sugges:Yifei Xuanfei Jiangzhuofang may function by raising cAMP,PKA,p-CREB expression in hippocampal neurons,activating the cAMP-PKA-CREB signaling pathway,promoting the formation of LTP,protecting the neurons,further improving learning and memory ability of VD rats.(2) Results of all the tests show that:Yifei Xuanfei Jiangzhuo fang has a better therapeutic effect on vascular dementia rats, suggest that the theory Treating vascular Dementia from the physiological function of the lung is feasible,and worth further exploring.更多还原