【作者】 沈峰；

【导师】 孙国杰；

【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2009， 博士

【摘要】 目的：老年性痴呆，又称阿尔茨海默病（AD），是发生于老年前期或老年期，以进行性记忆等认知障碍为主要症状，同时伴有精神行为异常的神经退行性疾病。其特征性临床表现为严重的学习记忆功能下降、分析判断能力衰退、情绪改变、行为失常、甚至意识模糊，严重影响了老年人的日常生活和社会活动能力，使其生命质量下降。世界人口正在加速变老。第五次人口普查表明，我国已经成为老龄国家。到本世纪中叶，我国60岁以上老年人将达4亿左右，届时每4个人中就有1个老年人。为此，包括我国在内的各国政府均加快了防治老年性痴呆的研究工作。而有效改善AD患者的学习记忆能力，提高其生存质量，是AD研究的重点和突破口所在。AD的发病机制复杂，许多信号分子在其中发挥重要作用，多个信号通路参与此过程，而临床上现有的针对AD发病的单基因或单靶向治疗药物并不能有效改善或控制患者症状，疗效短暂而局限，且副作用明显。针对单靶向药物对抗复杂疾病的缺点，近年来科学研究者提出并尝试将多靶向治疗策略应用于AD治疗。这是因为复杂疾病的发病往往由多种信号通路参与，而单靶向药物作用忽视了细胞内生物大分子之间的相互影响及信号通路中构成的网络系统的相互联系。并且有研究者认为，保护细胞膜和细胞内各种信号转导途径免受Aβ等的伤害，有可能成为改善AD患者认知功能和防治AD的突破。针灸以其疗效确切、作用持久、无副作用而受到医学界的普遍关注。近年来，针灸疗法在防治AD方面的临床和实验研究取得很大进展，对比发现针刺疗效不亚于中西药物，甚至在某些方面有优于西药的趋势。同时，针灸疗法所具有的多靶点、多途径、多层次整体治疗优势，也正符合现代医学针对复杂疾病提出的多靶向治疗策略。因此，深入研究针灸对AD的干预作用，对进一步理解衰老和针刺机制，推动临床研究具有重要意义。为此，本实验从学习记忆的相关中枢信号通路入手，进一步探讨针刺治疗AD的作用机制，为AD的针灸临床提供一定的实验依据。方法：将SD大鼠按随机数字表分为4组，即正常组、假手术组、模型组、电针组。采用双侧海马一次性注射聚集态Aβ_25-35制备AD大鼠模型，电针组选取双侧“肾俞”穴、双侧“内关”穴和“大椎”穴，接G6805-Ⅱ型电针治疗仪，选用2Hz连续波，强度为1mA，通电20min，每天1次，每周6次，共治疗2周。采用Morris水迷宫检测各组大鼠学习记忆能力的变化；采用HE染色，光镜下观察各组大鼠海马形态学的变化；采用分光光度法检测各组大鼠Glu的变化；采用放射免疫法检测各组大鼠海马cAMP的变化；采用免疫组化法检测各组大鼠海马PKA、CREB蛋白表达的变化；采用原位杂交法检测各组大鼠海马NMDAR1mRNA的变化。并进一步分析电针干预对上述各项指标的影响。结果：①定位航行实验中，假手术组与正常组相比，无明显差异（P＞0．05）；与假手术组比较，模型组大鼠的逃避潜伏期明显延长，差异有显著性（P＜0．01）；与模型组相比，电针组大鼠的学习记忆能力有明显的提高（P＜0．05）。②空间探索实验中，假手术组与正常组相比，无明显差异（P＞0．05）；与假手术组比较，模型组大鼠跨越平台的次数减少，差异有显著性（P＜0．01）；与模型组相比，电针组大鼠记忆平台的能力有明显提高（P＜0．05）。③HE染色：光镜下观察可见正常组和假手术组大鼠海马各区神经元染色清晰，呈圆形、椭圆形或锥形，排列整齐紧密。核呈圆形，大而清晰，核仁明显，形态完整。模型组大鼠海马注射针道附近可见细胞增生、聚集，核小深染，可能为胶质细胞；CA1区、CA₃区锥体细胞数量较假手术组明显减少，局部神经元缺失，残留的神经元排列松散不规则，多数细胞胞核固缩，核仁欠清晰，整个细胞深染成红色，部分呈现出多齿形、月牙型等凋亡形态，并伴有胶质细胞增多。电针组能明显改善上述神经元损伤，使海马神经细胞排列较整齐，核固缩现象改善。④分光光度法检测结果显示：模型组大鼠海马Glu含量明显高于假手术组，差异有显著性（P＜0．01）；与模型组相比，电针组大鼠海马Glu含量有明显的下降（P＜0．05）。⑤放射免疫法检测结果显示：模型组大鼠海马cAMP的含量明显降低，与假手术组比较有显著性差异（P＜0．01）；电针组大鼠海马cAMP含量与模型组比较有显著提高（P＜0．05）。⑥免疫组化法检测结果显示：模型组大鼠海马CA1、CA3区PKA的表达明显低于假手术组，差异有显著性（P＜0．01）；与模型组相比，电针组大鼠海马CA1、CA3区PKA表达明显增多（P＜0．05）。⑦免疫组化法检测结果显示：与假手术组比较，模型组大鼠海马CA1、CA3区CREB的表达明显降低，有显著性差异（P＜0．01）；电针组大鼠海马CA1区CREB的表达与模型组比较有显著提高（P＜0．05）。⑧原位杂交法检测结果显示：模型组大鼠海马CA1、CA3区NMDAR1mRNA阳性细胞平均灰度值与假手术组相比显著升高（P＜0．01）。电针组大鼠海马CA1、CA3区NMDAR1mRNA阳性细胞平均灰度值与模型组相比明显降低（P＜0．01）。结论：双侧海马一次性注射Aβ_25～35制备的大鼠模型成功的模拟了AD的行为学特征，海马神经细胞中Glu-NMDAR信号转导通路与cAMP-PKA-cREB信号转导通路参与了AD大鼠学习记忆障碍的过程，而电针“肾俞”穴、“内关”穴和“大椎”穴可以显著提高AD大鼠的学习记忆能力。其作用机制可能是通过以下途径实现的：通过降低Glu的神经毒性，保护海马神经细胞，并上调NMDAR1-mRNA的表达，充分活化NMDAR，改善Glu-NMDAR信号通路的传导；同时提高海马神经细胞cAMP、PKA及CREB的表达，激活其cAMP-PKA-CREB信号通路，从而加强LTP，改善学习记忆的能力。本实验也再次证明针刺具有多靶点、多途径、多层次、整体治疗的特点，能够有效的减轻AD大鼠海马神经细胞的损害，促使学习记忆相关信号通路正常转导，提高AD学习记忆能力。更多还原

【Abstract】 Objective: Alzheimer’s disease（AD） is a disease mainlydemonstrateted the symptoms of cognitive handicap of progressivehypomnesia occurred at the presensium or senectitude,which accompanied by psychological and dystropic neurodegene-rativesymptoms. Its characteristic clinical manifestations isa serious decline in learning and memory function, analyticaljudgement capacity of a recession, emotional changes, behavioraldisorders, and even clouding of consciousness, which hasseriously affected the daily lives and social activities ofolder people and led to a decline in the quality of their life.The world’s aging population is accelerating. The fifthcensus shows that our country has already become an agingcountry. Till the middle of this century, old people over 60years old will reach about 4 billion, in other words, there willbe one old people in every 4 at that time. As a result, eachcountry including ours has accelerated the research work ofAD’s prevention and treatment. Therefore, the keystone andbreakthrough of AD’s research consists in the effectiveimprovement of AD patients’ learning-memory ability and theirlife quality. The pathogenesis of AD is complicated. Many signalingmolecules have played an important role in it and some signalingpathways have taken part in the process. However, thesingle-gene or single-target therapeutic drugs for AD onclinical are not able to improve or control the patients’symptoms effectively, and also the therapeutic effect islimited and has obvious side effects. To deal with the disad-vantageof the single-target drugs towards the complex diseases,in these years, scientific researchers have presented and triedbringing multi-target treatment strategy into application ofAD’s therapy. The reason is that the complex diseases happeninghas usually involved various signaling pathways, while theeffect of single-target drugs have always ignored thebiological macromolecules’ interactions in cells and networksystem’s interconnections formed in the signaling pathways.And the researcher has indicated that protecting membrane andvarious signal transduction pathways from Aβand others’injury may be the breakthrough of promoting AD patients’cognitive function and AD’s prevention and treat-ment.Acupuncture gets widespread concern in the medicalprofession due to its exact effect, permanent effect and no sideeffects. In recent years, acupuncture therapy has got a greatimprovement in the clinical and experimental research of AD’sprevention and treatment. Comparison shows that acupuncture’stherapeutic effect is no worse than Chinese and westernmedicines, or even trend to be better than western medicinesin some part. At the same time, acupuncture therapy with itstherapeutic advantage of multi-target, multi-avenue and multi-levelhas also accorded with the multi-target therapeutic stra- tegy of modern medicine to the complex diseases. Therefore,making the deep research of acupuncture’s intervention effectto AD will make great senses to get further understanding ofaging and acupuncture’s mechanisms and consequently enhancethe clinical research.Therefore the experiment starts with the related centralsignal transduction of learning-memory to make the furtherexplore on the central mechanism of AD’s acupuncture treatment,which provides the experimental evidence for AD’s clinicaltreatment.Methods: SD rats were randomly divided into 4 groups: thenormal group, sham-operated group, model group, EA group.Bilateral hippocampus were injected condensed Aβ_25-35 to beprepared for rat model of AD. Electro-acupuncture group wereselected bilateral "Shenshu" points, bilateral "Neiguan"points and "Dazhui" points, then connected G6805-Ⅱtype ofelectroacupuncture therapeutic apparatus. We selected thefrequence of 2Hz continuous wave and the intensity of 1mA, last20min, every day one times, 6 times a week, a total of two weekstreatment. We used Morris water maze to test the changes inlearning and memory abilities in each group; used HE stainingto observe the group of morphological changes in rat hippo-campusby light microscope; used spectrophotometric method todetect the changes of Glu in each group; used radioimmunoassayto detect the hippocampal cAMP in each group changes; usedimmunohistochemistry to detect the expression changes of PKAand CREB protein in hippocampus in each group;and usedhybridization in situ to detect NMDAR1mRNA changes in hippo-campusin each group. And make a further analysis on the effect of electro-acupuncture’s intervention to every index above.Results:①Compared with the normal group, there was nosignificancein sham operation group in positioning navigationexperiment（P＞0.05）; compared with the sham operation group,the escape latency of rats in model group rats was longer thanthe former, there was a significant difference（P＜0.01）;Compared with model group, EA group learning and memory in ratssignificantly increased（P＜0.05）.②In space exploration experiments,there was no significance（P＞0.05） between sham-operatedgroup and normal group; compared with the sham operationgroup, the reduction of the number across the platform in modelgroup of rats was a significant difference（P＜0.01）; Comparedwith model group, the memory of rats in electroacupuncturegroup has improved, there was significance（P＜0.05）.③HEstaining: the neurons staining were clear, round, oval orconical, neatly arranged closely in normal group and shamoperation group rat hippocampal. Nuclear circular, large andclear, nucleolus obvious morphological integrity. In modelgroup, the number of rat hippocampal neurons decreasedsignificantly;the loss phenomenon were apparently, residualnerve cells arranged loosely and irregularly; the majority ofcell nuclei pyknosis, nucleolus unclear, Nissl body reduced ordisappeared, the whole deeply stained cells red, CAl area andCA3 area were markedly. Electro-acupuncture can obviouslyimprove the above-mentioned neuronal damage, so that increasethe number of hippocampal neurons, with a more tidy karyopy-knosisphenomenon improvement.④The testing result of spectropho-tometricshowed: Glu of model group’s hippocampus weresignificantly higher than the sham group, and there was a significant difference （P＜0.01）; Compared with model group,Glu of EA group’s hippocampus decreased significantly（P＜0.05）⑤The testing result of radioimmunoassay showed: Compared withsham-operated group, cAMP content of model group’s hippocampusdecreased significantly（P＜0.01）; Compared with the modelgroup,the cAMP content of EA group’s hippocampus significantlyenhanced（P＜0.05）.⑥The testing result of immunohistochemistryshowed: PKA’s expression of model group’s hippocampus wasobviously lower than sham-operated group, and the differencewas significant（P＜0.01）; Compared with model group, PKA’sexpression of electro-acupuncture obviously increased（P＜0.05）.⑦The testing result of immunohistochemistry showed: Comparedwith the sham-operated group, it was significant that CREB’sexpression of model group’s hippocampus decreased obviously（P＜0.01）; Compared with model group, CREB’s expression ofelectro-acupuncture group’s hippocampus enhanced significantly（P＜0.05）.⑧The testing result of hybridization showed:Compared with sham-operated group, it was significant thatNMDAR1mRNA’s expression of model group’s hippocampus decreasedobviously（P＜0.01）; Compared with the model group, NMDAR1 mRNA’s expression of electro-acupuncture group’s hippocampus increasedsignificantly（P＜0.01）.Conclusions: Bilateral hippocampal Aβ_25-35 one-time inject-tionof the preparation of rat model successfully simulates thebehavior characteristic, in hippocampal neurons, Glu-NMDARsignaling pathway and cAMP-PKA-cREB signaling pathway involvedin learning and memory impairment of AD process. And electro-acupuncture"Shenshu" point, "Neiguan" point and "Dazhui"point AD can significantly improve learning and memory in rats. Its mechanism may be achieved through the following means:Reduce Glu neurotoxicity, protect the hippocampal neurons andincrease the expression of NMDAR1-mRNA, make full activationof NMDAR, improve the Glu-NMDAR signaling pathway conduction,at the same time enhance cAMP, PKA and CREB’s expression ofhippocampal neurons, activate the cAMP-PKA-CREB signalingpathway in order to enhance LTP and improve the learning-memorycapacity. Acupuncture has proved once again that multi-target,multi-channel, multi-strata, the overall characteristics ofthe treatment can effectively alleviate the damage ofhippocampal neurons in AD rats so as to facilitate the signaltransduction pathway related to learning and memory normally,enhance the learning and memory of AD rats.更多还原