【作者】 董晓华；

【导师】 张丹参；

【作者基本信息】 河北医科大学 ， 药理学， 2012， 博士

【摘要】 阿尔茨海默病（Alzheimer’s disease，AD）是发生于老年期和老年前期的一种以进行性痴呆为特征的脑慢性退行性疾病，临床主要表现为记忆力减退、智能障碍及行为情绪等的异常改变。统计表明，65岁左右的老年人中AD发病率为10%，85岁以上的老年人中发病率为47%，AD患者的存活期预计可达到20年，但大多数病例在确诊后仅能存活8～10年，它已经成为人类的第四号杀手。随着人口老龄化进程加快，开发对AD有效的干预措施和治疗药物已成为AD研究的热点，也是一个倍受关注的社会问题。AD的发病机制一般包括胆碱功能降低，氧化应激，淀粉样肽的级联反应，炎症介质，甾体类激素减少。另外兴奋性毒性、细胞凋亡（apoptosis）、细胞周期调节异常也与AD关系密切。神经生化学揭示中枢氨基酸类神经递质与学习和记忆密切相关，Asp和Glu属于兴奋性氨基酸，Gly和GABA属于抑制性氨基酸，它们对维持神经系统的兴奋和抑制平衡起重要作用。AD时中枢各种氨基酸水平发生变化，尤其Glu和GABA间的平衡被破坏，是导致神经元损伤的关键因素。细胞凋亡是AD发生神经元退行性死亡的重要途径，因此干扰细胞凋亡可能成为治疗AD新方法。细胞凋亡受多种基因的调控，Bcl-2基因家族是很重要的调控基因，包括众多成员，其中Bax在凋亡中起促进作用，Bcl-2则拮抗细胞凋亡。Bcl-2/Bax组成一个平衡体系，Bax过剩细胞凋亡加速，Bcl-2过多则细胞凋亡被抑制。近年来，随着对细胞周期及其调控研究的深入，人们发现AD发病机制与细胞周期调控密切相关，提出了AD发病机制细胞周期假说：一旦细胞周期调控的某一环节出现异常，成年人中枢神经系统的神经元可再次进入细胞周期，但处在有丝分裂末期企图重新进入细胞周期的神经元将出现细胞周期停滞，最终形成AD某种病理特征或神经元凋亡。蛇床子素（osthole，Ost），7-甲氧基-8-异戊烯基-香豆素，传统中药蛇床子的组成成分，具有多种的药理作用，包括抗氧化、抗炎、抗肿瘤、抗血小板、雌激素样和抗疼痛等作用，目前已在临床上用于治疗皮肤病和性病多年。我们多年来一直从事蛇床子素改善学习记忆，抗衰老的研究，目前研究表明蛇床子素能通过抑制脑内乙酰胆碱酯酶（acetylcholinesterase，AChE）活性、增强谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）和超氧化物歧化酶（superoxide dismutase，SOD）活性而清除氧自由基（oxygen free radical，OFR），改善痴呆模型小鼠学习记忆障碍，且毒副作用小，能透过血脑屏障（blood-brain barrier，BBB），有望成为预防和治疗AD的有效药物。基于以上研究背景，本研究采用行为学实验和电生理学实验的方法，研究蛇床子素对Aβ25-35诱导AD大鼠认知障碍及中枢可塑性的影响；采用柱前衍生化高效液相色谱紫外检测法研究蛇床子素对Aβ25-35诱导的AD大鼠海马中氨基酸类神经递质水平变化；采用HE染色、TUNEL染色、免疫组织化学染色方法和流式细胞技术研究蛇床子素对Aβ25-35诱导的AD大鼠海马神经元凋亡、凋亡相关蛋白表达及细胞周期的影响。具体内容如下：1蛇床子素对AD大鼠学习记忆的影响目的：采用一次性侧脑室注射聚集态Aβ_25-35建立AD大鼠模型，并以不同剂量的蛇床子素进行干预，观察蛇床子素对AD模型大鼠认知障碍的影响。方法：SD大鼠随机分4组：生理盐水对照组（control group），模型组（model group），蛇床子素12.5mg·kg^-1组（Ost1group），蛇床子素25.0mg·kg^-1组（Ost2group）。实验d1，模型组和蛇床子素组大鼠侧脑室注射聚集态Aβ25-35建立AD大鼠模型，对照组大鼠注射生理盐水。造模后对照组和模型组大鼠腹腔注射（peritoneal injection，ip）溶剂1mL·kg^-1，蛇床子素组大鼠分别给予不同剂量蛇床子素，所有动物都连续给药14d。造模后d10-d14进行Morris水迷宫（morris water maze，MWM）实验，大鼠训练4d（d1-d4），记录动物找到站台的时间（逃避潜伏期）。最后一天训练后24h（d5），移走站台进行测试实验，记录大鼠在目标象限停留时间和穿过站台位点的次数。结果：随着训练天数的增加，各实验组潜伏期均不断缩短；d1各组间潜伏期相比无差异；模型组与对照组相比，潜伏期从实验d2起延长，目标象限搜寻时间百分率和穿过站台次数减少；蛇床子素组与模型组相比，d2起潜伏期明显缩短，目标象限搜寻时间百分率和穿过站台次数增加。小结：一次性侧脑室注射聚集态Aβ_25-35，成功建立AD大鼠模型，出现空间学习记忆能力减退，蛇床子素具有改善AD大鼠学习记忆障碍的作用。2蛇床子素对AD大鼠海马HFS诱导LTP的影响目的：采用一次性侧脑室注射聚集态Aβ_25-35建立AD大鼠模型，并以不同剂量的蛇床子素进行干预，观察蛇床子素对AD模型大鼠中枢突触可塑性的影响。方法：大鼠造模d14，参照Pellegrino大鼠脑立体定位图谱中的定位方法定位刺激电极和记录电极，采用在体细胞外记录LTP的电生理学方法，观察蛇床子素对AD大鼠海马高频刺激（high-frequency stimulation，HFS）诱导的长时程增强（long-term potentiation, LTP）现象的影响。结果：各组在给予HFS后，PS幅值在观察的60min内明显增加，并形成LTP现象，但每组HFS后PS幅值增长的幅度不同；与对照组相比，模型组在HFS后60min内各时间点PS幅值减少；而蛇床子素治疗组较模型组PS幅值在高频刺激后的每个时间点升高。小结：AD模型大鼠海马出现HFS诱导LTP抑制，蛇床子素增强海马神经元的突触可塑性，逆转AD大鼠海马HFS诱导LTP的抑制，从细胞突触水平表明蛇床子素具有改善AD大鼠学习记忆障碍的作用。3蛇床子素对AD大鼠海马氨基酸水平的影响目的：观察AD大鼠海马氨基酸类神经递质水平变化，从中枢Glu/GABA学习记忆调节系统探讨蛇床子素改善AD大鼠学习记忆障碍的作用机制。方法：大鼠造模d14，采用柱前衍生化高效液相色谱法测定Asp, Glu,Gly和GABA含量，紫外检测波长是360nm。麻醉状态下脱臼处死大鼠，立即冰上分离海马并用甲醇和水（体积比1:1）制成10%匀浆，4℃离心10min （10000rpm），留存上清在-80℃冰箱用于测定氨基酸含量。测定前将标本复溶，以10000rpm低温高速离心10min，取上清200μL；加入200μL乙腈，混匀，10000rpm低温高速离心10min，沉淀蛋白；取上清液+200μL碳酸氢钠+100μL2,4-二硝基氟苯（DNFB），混匀；水浴60℃，暗中衍生1h；过滤后进样20μL，测定氨基酸含量。结果：与对照组比较，模型组大鼠海马中Asp、Glu、Gly和GABA含量及Glu/GABA比值显著升高，4种氨基酸中谷氨酸升高最为明显；与模型组比较，蛇床子素各剂量组Glu含量显著降低，Asp、Gly和GABA含量下降，但不具有统计学意义，蛇床子素各剂量组Glu/GABA比值较模型组显著降低，但都高于对照组。小结：一次性侧脑室注射Aβ_25–35建立AD大鼠模型，可导致大鼠海马内氨基酸类递质代谢紊乱，尤其是Glu水平和Glu/GABA比值显著升高，这将导致兴奋性毒性。蛇床子素可通过影响Glu/GABA学习记忆调控系统而抑制Glu的兴奋性毒性，对中枢神经元具有保护作用。4蛇床子素对AD大鼠海马神经元凋亡的影响目的：采用一次性侧脑室注射聚集态Aβ_25-35建立AD大鼠模型，并以不同剂量的蛇床子素进行干预，观察蛇床子素对AD大鼠海马细胞凋亡、凋亡相关蛋白表达及细胞周期的影响。方法：大鼠造模d14麻醉，肝素化生理盐水及4%多聚甲醛进行心脏灌注固定30min，快速断头取脑，切成厚度约3mm的组织块，4%多聚甲醛固定24h。然后进行脱水、透明、浸蜡、包埋、切片、贴片，分别进行苏木素-伊红染色、Bcl-2和Bax蛋白免疫组化法染色、TUNEL法染色。在光镜下观察切片并记录结果。另一部分大鼠在造模后d14断头处死取海马组织，制备单细胞悬液，70%预冷乙醇固定，4℃放置24h以上。50μg·mL^-1碘化丙啶液4℃衍生30min，流式细胞仪分析细胞周期。结果：①HE染色：对照组光镜下未见有明显病理改变；模型组海马神经细胞排列紊乱，部分神经细胞出现凋亡征象，视野内可见凋亡细胞；蛇床子素组海马结构接近对照组，凋亡细胞较模型组明显减少。②TUNEL法检测凋亡细胞：对照组极少见凋亡细胞；模型组视野内凋亡细胞与对照组比较明显增多；蛇床子素组与模型组比较，凋亡细胞数减少。③Bcl-2、Bax蛋白免疫组化法检测：对照组有极少量Bcl-2、Bax蛋白表达，与对照组比较，模型组Bcl-2，Bax蛋白表达都升高，Bax蛋白表达升高幅度更大，因此Bcl-2/Bax蛋白比值降低。蛇床子素组与模型组比较，Bcl-2蛋白表达增多，Bax蛋白表达减少，Bcl-2/Bax蛋白比值升高。④流式细胞仪检测细胞周期：模型组与对照组比较，模型组G₀/G₁期百分率升高，S期百分率降低，G₂/M期百分率升高，增值指数（proliferation index，PI）降低。蛇床子素治疗组与模型组比较，S期百分率和PI均显著升高，而G₂/M期百分率降低。小结：蛇床子素调节AD大鼠海马凋亡相关蛋白Bcl-2和Bax，升高Bcl-2/Bax蛋白比值，具有抗凋亡、保护海马神经元的作用。另外蛇床子素增加S期细胞百分率，促进G₂/M期细胞进一步分裂，增强细胞增殖活性，调节细胞周期，有利于维持海马正常的生理功能。结论：1本研究采用一次性侧脑室注射聚集态Aβ25-35，成功建立AD大鼠模型。模型大鼠出现AD征象：学习记忆能力减退；HFS诱导海马LTP的PS幅值降低；海马凋亡细胞增多；海马细胞增殖活性下降，细胞阻滞在G₂/M期；海马内Glu水平及Glu/GABA比值升高，产生兴奋性毒性。2蛇床子素改善AD大鼠学习记忆障碍，增强海马神经元的突触可塑性，从整体动物水平（行为学）及细胞突触水平（海马LTP）表明蛇床子素具有改善AD大鼠认知障碍的作用。3蛇床子素降低AD大鼠海马内Glu水平及Glu/GABA比值，调节Glu/GABA学习记忆调控系统，减弱Glu的兴奋性毒性，可能是其改善AD大鼠学习记忆障碍的作用机制之一。4蛇床子素通过调节AD大鼠海马凋亡相关蛋白Bcl-2和Bax，升高Bcl-2/Bax蛋白比值，具有抗凋亡、保护海马神经元的作用，可能是其改善AD大鼠学习记忆障碍的作用机制之一。5蛇床子素通过增加AD大鼠海马S期细胞百分率，促进G2/M期细胞进一步分裂，增强细胞增殖活性，调节细胞周期，有利于维持海马正常的生理功能，可能是其改善AD大鼠学习记忆障碍的作用机制之一。更多还原

【Abstract】 Alzhe mer’s disease （AD） is chronic degenerative brain diseasecharacterized by progressive dementia and the main clinical manifestationsinclude memory loss, mental retardation and abnormal changes in behaviorand mood. According to the statistics, the incidence of AD is about10%incrowd of about65years old and rises up to47%in the population over85years of age. The survival time of patients with AD is expected to reach20years, but most of the cases after diagnosis can only survive8to10years, soAD has become the fourth killer to human’s health.With rapid aging of thepopulation, the development of effective interventions and medications to cureAD has become a hot issure in AD research，and also an anxious socialproblem.The proposed pathogenic mechanisms of AD generally include loss ofcholinergic function, oxidative stress, amyloid cascade, inflammatorymediators and steroid hormone deficiencies. In addition, excitotoxicity,apoptosis and dysregulation of cell cycle are in close relation to AD.Neurobiochemistry revealed that central amino acid neurotransmitters are inclose relaiton to learning and memory. Asp and Glu are excitatory amino acids,Gly and GABA are inhibitory amino acids and they are very import in thebalance of exitation and repression of the nervous system.The level of aminoacids in the centre becomes abnormal when AD happens, especially thebalance between Glu and GABA, which is the key point of neuron injury.Apoptosis is a significant path of neuron retrograde death, so the interferenceto apoptosis could be a possible new method to treatment of AD. Apoptosis isregulated by many kinds of genes and Bcl-2gene family is very importantcontrolling gene containing numerous members in which Bax promotesapoptosis and Bcl-2restrains apoptosis. Bcl-2/Bax is a balanced system— while Bax is redundant the apoptosis process is promoted and while Bcl-2isredundant the process is restrained. In recent years, with the deep study of cellcycle and the regulation, people found that the mechanism of AD is in relationto the regulation of cell cycle and proposed the cell cycle hypothesis about themechanism of AD. The adult neurons of central nervous system can enter cellcycle again when some component elements become abnormal in theregulation of the cell cycle, but neurons in mitosis anaphase will appear thefailure of cell division and cell cycle arrest finally and form some pathologicalfeatures of AD or neuron apoptosis.Osthole （7-methoxy-8-isopentenoxy-coumarin） is an ingredient oftraditional Chinese medicine （TCM） from natural product Cnidium monnieri（L.） Cusson, possesses a variety of pharmacological properties, includingantiosteoporotic, antihepatitic, antiallergic, antiseizure and antiproliferativefunctions and so on, and has been clinically used in the treatment of skindisease and gynecopathy for many years.We have engaged in the research ofosthole on improvement of learning and memory and anti-aging for manyyears.The previous studies showed that osthole can improve learning andmemory impairment in dementia model mouse via inhibitingacetylcholinesterase （AChE） activity or by enhancing the activities ofglutathione peroxidase （GSH-PX） and superoxide dismutase （SOD） andclearing oxygen free radical （OFR） in brain. Osthole can pass through theblood-brain barrier and its side effects are small, so it is expected to becomethe effective drug to prevent and treat AD.Based on these profiles,the study investigated the effects of osthole oncognitive impairment and central synaptic plasticity of AD rats induced byAβ25-35via behavioral and electrophysiological experiment method,observedthe effects of osthole on contents of amino acids in hippocampus of AD ratsinduced by Aβ25-35via pre-column derivatization high performance liquidchromatography and researched the effects of osthole on neuronal apoptosis,protein expression related to apoptosis and cell cycle in hippocampus of ADrats induced by Aβ25-35via HE, TUNEL and immunohistochemical staining method and flow cytometry. The specific contents as follows:1Effects of osthole on learning and memory of AD ratsObjective: We aimed to investigate the effects of osthole on cognitiveimpairment of AD rats induced by once intracerebroventricular （i.c.v.）injection of Aβ_25-35.Methods: SD rats were randomly assigned to control, model, low-doseosthole (12.5mg·kg^-1, Ost1), and high-dose osthole (25.0mg·kg^-1, Ost2)group. On the first day of the experiment, an intracerebroventricular （i.c.v.）injection of Aβ25-35was administrated to establish AD rat model. After i.c.v.injection, control and model group rats were intraperitoneally injected withsolvent1mL·kg^-1, and osthole treatment group rats were intraperitoneallyadministered with osthole. All animals were administered continuously for14days. Morris water maze test was performed from day10to14after injectionof Aβ_25-35. Rats were trained four days （d1-d4） and the escape latency wasrecorded. Twenty-four hours after the last training day （d5）, a probe trial wasmade under the condition of removing the platform and the residence time inthe target quadrant and the number of crossing the platform site wererecorded.Results: With the increase of training day, the escape latenciescontinuously reduced in each experimental group, and there was no differencebetween the groups on the first day of training. Compared with control group,model group displayed longer escape latency from d2and lower percentage oftarget quadrant searching time and fewer number of crossing platform （inprobe trial）. Compared with model group, osthole groups showed shorterescape latency fron d2and higher percentage of target quadrant searching timeand more number of crossing platform.Summary: The study indicated the establishment of AD rat model byonce i.c.v. injection of Aβ_25-35is successful. The model rats showed learningand memory decline and osthole improved the learning and memoryimpairment of AD rats.2Effects of osthole on hippocampal LTP induced by HFS of AD rats Objective: We aimed to investigate the effects of osthole on centralsynaptic plasticity of AD rats induced by once intracerebroventricular （i.c.v.）injection of Aβ_25-35.Methods: Electrophysiological test was conducted on day14after modelestablishment. According to Pellegrino stereotaxic atlas of rat brain,positioned stimulating and recording electrode. We adoptedelectrophysiological methods of extracellular recording in vivo to observe theeffects of osthole on hippocampal long-term potentiation （LTP） phenomenoninduced by high frequency stimulus （HFS） of AD rats induced by onceintracerebroventricular （i.c.v.） injection of Aβ_25-35.Results: In all groups, the PS amplitude obviously increased during the60min observation and the LTP phenomenon was induced by HFS, but thegrowth extent of PS amplitude was different in each group. The PS amplitudein model group was lower than that in control group, while the PS amplitudein osthole groups was higher than that in model group on each time point afterHFS.Summary: Our experiments discovered that AD model rats appeared theinhibitory action of LTP induced by HFS, osthole could enhance neuronalsynaptic plasticity in the hippocampus and reversed the inhibitory action ofLTP induced by HFS of AD rats, which indicated osthole could improvelearning and memory impairment of AD rats from cell synaptic level.3Effects of osthole on level of hippocampal amino acids of AD ratsObjective: We aimed to observe the effects of osthole on level ofhippocampal amino acids of AD rats and investigate the mechanism of ostholeon cognitive impairment of AD rats from Glu/GABA learning and memoryregulatory system.Methods: Amino acids determination was conducted on day14afterestablishment of AD model.The levels of Asp, Glu, Gly and GABA weredetermined by pre-column derivatization high performance liquidchromatography （HPLC） and the wavelength of the ultraviolet detection was360nm. Rats were executed after anesthesia and the hippocampus tissues were separated on ice. Made10%homogenate with methanol and distilled water（1:1V/V） and centrifuged with10000rpm speed for10min. The supernatantswere stored at-80℃.The specimens were dissolved before determination andcentrifuged with10000rpm speed for10min at low temperature. Acetonitrile200μL and supernatant200μL were mixed and centrifuged with10000rpmspeed for10min at low temperature to precipitate protein. The supernatant,sodium hydrogen carbonate200μL and2,4-dinitrofluorobenzene100μL weremixed and derived for1h in60℃water bath. The whole process should payattention to avoid light. Finally the sample was filtered and taken out20μLliquid to determine the contents of amino acids.Results: Compared with control group, the contents of Asp, Glu, Gly,GABA and the ratio of Glu/GABA obviously increased in model group. Theincrease extent of Glu was more than those of other amino acids. Comparedwith model group, the contents of Glu in osthole groups decreased withsignificant difference, and the contents of other amino acids decreased withoutsignificant difference.The ratios of Glu/GABA in osthole groups decreasedcompared with that in model group, while higher than that in control group.Summary: HPLC results showed that the establishment of AD rat modelby i.c.v. injection of Aβ_25-35could give rise to the metabolic disturbance ofamino acid transmitters in hippocampus and the increasion of Glu content andGlu/GABA ratio, which resulted in excitotoxicity. Osthole could inhibit theexcitotoxicity of Glu by regulate the Glu/GABA learning and memoryregulatory system and possessed the protective action to central neurons.4Effects of osthole on neuronal apoptosis of AD ratsObjective: We aimed to investigate the effects of osthole onhippocampal neuronal apoptosis, protein expression related to apoptosis andcell cycle of AD rats.Methods: Rats were anesthetized on day14after establishment of ADmodel and perfused heart with heparinized normal saline and4%paraformaldehyde for30minutes. Decapitation and brain extraction wereperformed quickly.Cut the brain into pieces of3mm and fixed the tissues with 4%paraformaldehyde for24hours. Dehydration, lucidification, steeping wax,embedding, slicing and coating were performed in order andhematoxylin-eosine staining, Bcl-2and Bax immunohistochemistry stainingand TUNEL staining were also performed respectively. Slides were observedwith light microscope and results were recorded. Another part of rats wereexecuted to obtain hippocampus tissues on day14after model establishment.Made the monoplast suspension and fixed them with70%precooling alcoholfor more than24hours at4℃. Derived with propidium iodide of50μg·mL-1for30minutes at4℃and analyzed the cell cycle with flow cytometry.Results：①HE staining: No remarkable neuronal abnormalities in thehippocampus of the control group rats were observed, while the slides ofmodel group showed the disorder of neurons array, some neurons appearedapoptosis signs and some apoptotic cells was observed in vision. While theneurons in the osthole group were close to those of control group and thenumber of apoptotic cells was decreased compared to the number in modelgroup.②Apoptosis detected with TUNEL: There were few apoptotic cells inthe control group and much more apoptotic cells in the model group than thosein the control group. There were fewer apoptotic cells in the osthole groupthan those in the model group.③Bcl-2and Bax detected withimmunohistochemistry: There were an extremely small quantitiy of Bcl-2andBax positive cells in the control group. Compared with the control group, BothBcl-2and Bax expression increased, the Bax expression increased much morethan Bcl-2expression and so Bcl-2/Bax ratio decreased in the model group.Compared with the model group, Bcl-2expression increased, Bax expressiondecreased and Bcl-2/Bax ratio increased in the osthole group.④Cell cycledetected with flow cytometry: Compared with the control group,the G0/G1stage percentage increased, the S stage percentage decreased, the G₂/M stagepercentage increased and proliferation index decreased in the modelgroup.Compared with the model group, both the S stage percentage and PIincreased and the G₂/M stage percentage decreased in the osthole groups.Summary: Osthole could increase the Bcl-2/Bax ratio by regulating Bcl-2and Bax, proteins related to apoptosis, inhibit apoptosis, and protecthippocampus neurons. Moreover, osthole could increase the percentage of Sphase cells, promote the G₂/M phase cells to divide further, strengthen theproliferation activity of cells, regulate cell cycle and benefit to themaintenance of normal physiological function of hippocampus.Conclusions:1The study constructed the AD rat model establiahment via once i.c.v.injection of Aβ_25-35is successful.The model rats showed AD signs: learningand memory impairment, decrease of the PS amplitude of hippocampalLTP induced by HFS, increase of hippocampal apoptosis cells, decrease ofthe proliferation activity of hippocampus cells, cell retardation at G₂/Mphase, increase of Glu and Glu/GABA ratio leading to excitotoxicity.2Osthole can improve the learning and memory impairment and strenghenthe synaptic plasticity in hippocampus of AD rats, which indicated thatosthole has the effect of improving the cognitive disorder of AD ratsimproved from the level of whole animal and cell synapsis.3Osthole can cut down the level of Glu and the Glu/GABA ratio inhippocampus of AD rats, regulate the Glu/GABA learning and memoryregulatory system, and decrease the excitotoxicity of Glu, which may beits one of the mechanisms of improving learning and memory impairmentof AD rats.4Osthole can increase the Bcl-2/Bax ratio by regulating Bcl-2and Bax,proteins related to apoptosis, inhibit apoptosis and protect hippocampusneurons, which may be its one of the mechanisms of improving learningand memory disorder of AD rats.5Osthole can regulate cell cycle and benefit to the maintenance of normalphysiological function of hippocampus by increasing the percentage of Sphase cells, promoting the G₂/M phase cells to divide further andstrengthening the proliferation activity of cells, which may be its one ofthe mechanisms of improving learning and memory disturbance of ADrats.更多还原