【作者】 万博鹏；

【导师】 孙国杰；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的阿尔茨海默病(Alzheimer’s disease，AD)，俗称老年性痴呆，是一种常见的中枢神经系统退行性疾病，是老年人脑功能退化的一种表现，以进行性、不可逆性出现智能减退为主要临床特征，包括近期记忆、思维、认知、定向、理解、计算、判断能力等功能的减退和不同程度的人格异常改变。随着全球人口老龄化的加重，AD已经成为人类继心脏病、癌症和脑卒中之后第4位致死原因。AD患者晚期劳动能力丧失，生活不能自理，心理适应能力亦急剧下降，给家庭和社会造成沉重治疗负担。本课题采用双侧海马一次性注射聚集态Aβ25-35造成AD大鼠模型，运用艾条温和灸“肾俞”、“足三里”、“百会”进行治疗，从JNK信号通路介导的细胞凋亡反应的角度探讨AD模型大鼠脑内神经元损伤机制及艾灸干预的作用机制。方法健康雄性SD大鼠（15月龄）50只，体重(400±50)g，由华中科技大学同济医学院实验动物学部提供。提前1周将大鼠运到实验室适应性喂养，安静清洁环境下定时给以饮水及饲料。然后进行Y型水迷宫筛选，淘汰反应过于迟钝或特别敏感的大鼠，将合格大鼠按随机数字表法分为4组，每组10只，分别为正常组、假手术组、模型组、艾灸组。采用双侧海马一次性注射聚集态Aβ25-35造成AD大鼠模型，造模完成后第2日开始进行艾灸治疗，在大鼠“肾俞”、“足三里”、“百会”穴上方2～3cm处温和灸。每日治疗1次，每穴持续艾灸5min，6日为1疗程，疗程间休息1日，共3个疗程。Morris水迷宫法测定其学习记忆能力的行为学变化，HE染色及透射电镜观察大鼠海马CA1区神经细胞结构的形态学变化，免疫组化SP法观察艾灸对AD模型大鼠海马区磷酸化JNK蛋白(p-JNK)、神经细胞凋亡的关键调控因子Bcl-2、Bax、Caspase-3表达的影响。全部数据输入计算机，用SPSS16.0统计软件包进行处理，所得数据以均数±标准差（x s）表示。结果（1）Morris水迷宫行为学试验发现，各组大鼠逃避潜伏期随着游泳天数的增加而逐渐缩短，但各组之间表现出的学习记忆能力有很大差异。大鼠双侧海马一次性注射Aβ25-35聚集液后，定位航行试验中，模型组大鼠5天平均潜伏期及后3天平均潜伏期均较其他组明显延长，大鼠在实验平台象限的游泳距离占游泳总距离百分比(象限百分比)明显减少；空间探索试验中，模型组大鼠跨越原平台位置的次数比其他各组明显减少，大鼠在实验平台象限的游泳距离占游泳总距离百分比(象限百分比)亦明显减少，与正常组及假手术组比较，有显著性差异(P<0.01)。说明模型组大鼠学习记忆能力受到严重损害，双侧海马一次性注射Aβ25-35制备AD模型成功。对AD大鼠采用艾灸治疗后，定位航行试验中，大鼠5天平均潜伏期和后3天平均潜伏期均明显缩短；空间探索试验中，大鼠跨越原平台位置的次数明显增多；在实验平台象限的游泳距离占游泳总距离百分比(象限百分比)明显增高，与模型组比较，经统计学分析，有显著性差异(P<0.01)。说明艾灸对AD模型大鼠的学习记忆功能具有明显改善作用，艾灸疗法是治疗学习认知功能障碍的有效方法之一，可广泛运用于临床。（2）HE染色观察结果显示，模型组大鼠海马CA1区神经细胞数量较正常组和假手术组明显减少，神经细胞受损缺失，细胞核固缩，核仁欠清晰，部分神经细胞呈现出月牙型、多齿形等凋亡形态。运用艾条温和灸“肾俞”、“足三里”、“百会”治疗后，大鼠海马CA1区神经细胞核固缩现象明显改善，形态较规则，排列较整齐。透射电镜观察结果显示，Aβ在大鼠脑内能引起海马CA1区神经细胞的退行性改变，神经元体积缩小，细胞核形态不规则，细胞浆和细胞器密度增高，细胞器结构受损，显示不清，线粒体肿胀，基质清淡、有空泡，数目减少，还可见到线粒体嵴呈畸形样改变，内质网扩张，脂褐素沉积增多。运用艾灸治疗后，大鼠海马CA1区神经细胞水肿较模型组明显减轻，内质网扩张及线粒体肿胀明显改善，高尔基体、线粒体、核糖体数目较模型组明显增多。表明艾灸治疗可抑制注射Aβ导致的大鼠海马区神经细胞损伤变性，促进神经细胞的修复，艾灸对AD模型大鼠海马神经细胞的退行性改变有明显改善作用。（3）模型组大鼠海马区p-JNK、Bax、Caspase-3阳性神经元计数较正常组、假手术组显著升高，Bcl-2阳性神经元计数则较正常组、假手术组显著降低，经统计学分析，均具有显著性差异（P<0.01）；艾灸组大鼠海马区p-JNK、Bax、Caspase-3较模型组表达明显下降，Bcl-2阳性神经元计数则较模型组明显升高，经统计学分析，均具有显著性差异（P<0.01）。结论（1）海马区注射Aβ25-35可以激活JNK信号转导途径，JNK的激活导致促凋亡蛋白Bax释放增多，抗凋亡蛋白Bcl-2释放减少，引起了Caspase级联反应，进而活化细胞凋亡的关键蛋白酶Caspase-3，启动了依赖Caspase通路的凋亡程序，从而诱导AD大鼠脑内神经元的退行性改变，引起大鼠学习记忆障碍。（2）艾灸治疗可引起p-JNK、Bax、Caspase-3在大鼠海马区的表达明显减少，Bcl-2的表达明显增多，可抑制注射Aβ导致的大鼠海马区神经细胞损伤变性，促进神经细胞的修复，艾灸对AD模型大鼠海马神经细胞的退行性改变有明显改善作用。（3）艾灸治疗AD的作用机制，可能是通过抑制JNK信号通路，减轻促凋亡蛋白Bax、Caspase-3的释放,促进抗凋亡蛋白Bcl-2的产生，从而抑制脑内神经细胞的凋亡,减少神经元的变性丢失，进而改善Aβ所致的大鼠学习记忆障碍。更多还原

【Abstract】 ObjectiveAlzheimer’s disease(AD) is a kind of degenerative disease incentral nervous system with clinical manifestation of progressivedecrease of learning and cognition ability, decline in recentmemory， thinking， perception, orientation, comprehension，calculation， judgment function and some abnormal personalitychanges. AD has become the fourth cause of death besides heartdisease，tumor and apoplexy in the aged people. The loss of advancedlabor ability, the sharp decline of self-care ability and mentalability in AD patients, caused a heavy burden to the family andsocial treatment. AD morbidity has become a serious public healthproblem because of its rapid increase tendency with nationpopulation aging. This study is designed to research on the actionmechanism of cell apoptosis mediated by JNK signal pathway intreating the rats with Alzheimer disease by moxibustion. The ADmodel was established by stereotaxis injecting agglutinated Aβ25-35into rat’s bilateral hippocampus. The rats with AD weretreated by moxibustion at the points of “Shenshu”、“Zusanli”and“Baihui”. MethodsFifty normal male SD rats with15months old, weighing400±50g were selected. Firstly they were detected with the method ofY type water maze to eliminate the rats with too clumsy or toosensitive reactions. The qualified rats were randomly divided into4groups with10cases in each group, respectively normal group,sham-operation group, model group and moxibustion group. AD modelswere established by stereotaxis injecting agglutinated Aβ25-35intorat’s bilateral hippocampus. Then AD model rats were treated bymoxibustion at the points of“Shenshu”、“Zusanli” and “Baihui”(left and right “Shenshu”,“Zusanli” respectively alternativeuse every other day)for3therapy courses，once daily with6daysa course， moxibustion for5minutes at each point, and one dayrest between two courses. The learning and memory ability wasdetected by Morris water maze test, morphological changes inhippocampal CA1region of rat neural cell structure were observedby HE staining and transmission electron microscope, the expressionchanges of p-JNK、Bcl-2、Bax、Caspase-3of hippocampus zone in ratswere observed by immunohistochemical method of SP. All data wereput into the computer and analyzed by SPSS16.0statistical package.The experiment results were expressed as mean±standard deviation（x s）.Results（1）From the Morris water maze test, we found the escape latencygradually shortened in each group as the swimming days increased,but he learning and memory ability between the groups showeddifferently. After stereotaxis injecting agglutinated Aβ25-35into rat’s bilateral hippocampus, the average escape latency of five daysand the last three days significantly lengthened, and the timesacross the platform position decreased remarkably in model group.In the Place navigation test and spatial probe test, the quadrantpercentage of swimming distance in the platform quadrant accountingfor the total swimming distance remarkably lowered in model group.There were remarkable differences compared with normal group andsham-operation group (P<0.01).The results indicated the rat’sfunction of learning and memory seriously damaged in model group.After the treatment by moxibustion, the average escape latency offive days and the last three days shortened obviously, and the timesacross the platform position significantly increased, and thequadrant percentage of swimming distance in the platform quadrantaccounting for the total swimming distance remarkably heightenedin the rats of moxibustion group. There were significantdifferences compared with model group (P<0.01). The results showedmoxibustion was effective in preventing the learning and memorydysfunction.（2）HE staining results showed that in model group, the numberof nerve cells in the hippocampus CA1zone reduced significantlycompared with normal group and sham-operation group. Nerve cellsdamaged with pyknotic nuclei, and nucleoli was not clear. Part ofthe nerve cells showed crescent type, multi tooth shaped apoptoticmorphology. After the treatment by moxibustion, nervouskaryopyknosis phenomenon was obviously improved with regular shape,and arranged more neatly. Transmission electron microscope showedsome nerve cells in the hippocampus CA1zone of the rats in modelgroup degenerated, the volume of nerve cell contracted with irregular shape. Electron density in cytoplasm and organelleobviously enhanced. Nerve cell structures were damaged anddisplayed unclearly. Mitochondrion swelled, even mitochondrialcrista was abnormal and deformed. Endoplasmic reticulum of interorganelle expanded and the deposition of lipofuscin increased.After the treatment by moxibustion, the edema of nerve cellssignificantly reduced, dilation of endoplasmic reticulum andmitochondria swelling significantly improved, golgi bodies,mitochondria, ribosomes obviously increased in comparison with themodel group. The experiment indicated that moxibustion hadsignificant effect in ameliorating the degenerative hippocampalneural cells change.（3）In model group, the number of p-JNK、Bax、Caspase-3positiveneurons in the rat hippocampus region obviously increased, and thenumber of Bcl-2positive neurons significantly decreased. Therewere remarkable differences compared with normal group andsham-operation group (P<0.01).In moxibustion group, the number ofp-JNK、Bax、Caspase-3positive neurons in the rat hippocampus regionobviously lowered, and the number of Bcl-2positive neuronssignificantly heightened. There were remarkable differencescompared with model group (P<0.01).Conclusion（1） Injecting agglutinated A β25-35into the bilateralhippocampus can activate the JNK signal pathway. The activation ofJNK leads to the increased release of Pro apoptotic protein Bax,and the decreased release of the anti apoptotic protein Bcl-2,furtherly, causing the Caspase cascade and activating the releaseof key enzyme of apoptosis Caspase-3. Then the degenerative neurons change induced in the brain of AD rats with learning and memoryimpairment.（2）The treatment by moxibustion can decrease the number ofp-JNK、Bax、Caspase-3positive neurons, and increase the number ofBcl-2positive neurons in the rat hippocampus region, so as torestrain the injury of neural cell degeneration caused by injectionof Aβ, promote the recovery of nerve cells and obviously amelioratethe degenerative hippocampal neural cells change.（3）Restraining the JNK signal pathway, relieving the releaseof Pro apoptotic protein Bax、Caspase-3, and promoting the releaseof the anti apoptotic protein Bcl-2, so as to inhibit the apoptosisof nerve cells, protect and recover the brain neurons and finallyimprove the learning and cognition ability, which may be the actionmechanism of treating AD by moxibustion.更多还原