【作者】 黄忠仕；

【导师】 黄仁彬；

【作者基本信息】 广西医科大学 ， 药理学， 2008， 博士

【摘要】 阿尔茨海默症（Alzheimer’s disease,AD）又称老年痴呆症,是一多发在老年,以近期记忆障碍为主要临床症状,以老年斑（SP）、神经元纤维缠结（NFT）和神经元丢失为主要病理改变的进行性神经变性疾病,严重影响患者的认知功能、记忆功能、语言功能、视空功能、社会生活能力、个人生活能力和情感人格等,其病因和发病机制尚不清楚。目前受到广泛认同的病因和机制主要有以下几种学说:神经递质缺陷;钙稳态失调;自由基损伤;基因突变;能量代谢障碍;神经细胞凋亡;兴奋性递质毒性;淀粉样蛋白（AB）沉积等。龙眼参[Millettia pulchra Kurz var laxior（Dunn）Z.Wei,LYS]是广西壮族习用药材,有补气、补血、提高免疫力、抗衰老、抗应激等作用,可强身健体、消除疲劳,主要用于老年健忘、小儿智力低下等疾病的治疗,亦用于体弱多病及病后、产后虚弱等的滋补。本研究从龙眼参提取多糖成分,并对龙眼参多糖[Millettia pulchra Kurz var laxior（Dunn）Z.Wei polysaccharides,LYSP]抗痴呆作用及机制进行了研究。实验采用AB25-35诱导NG108-15细胞作为痴呆细胞模型,采用昆明种小鼠皮下注射东莨菪碱或D-半乳糖、或灌胃40%乙醇以及采用快速老化小白鼠（SAM）等建立各种痴呆动物模型。昆明种小鼠实验的分组及给药为:①正常对照组:生理盐水,30ml·kg^-1;②模型组:生理盐水,30ml·kg^-1;③脑复康组:脑复康0.5g·kg^-1;④多糖低剂量组:龙眼参多糖45mg·kg^-1;⑤多糖中剂量组:龙眼参多糖90mg·kg^-1;⑥多糖高剂量:龙眼参多糖180mg·kg^-1。治疗结束后观察龙眼参多糖对各种痴呆小鼠Y型水迷宫正确次数、血清和脑组织Ach和AchE活性的影响。SAM小鼠实验的分组及给药为:①SAMR1对照组:生理盐水,30ml·kg^-1;②SAMP8空白组:生理盐水,30ml·kg^-1;③阳性药对照组:石杉碱甲0.02mg·kg^-1;④多糖低剂量组:龙眼参多糖45mg·kg^-1;⑤多糖中剂量组:龙眼参多糖90mg·kg^-1;⑥多糖高剂量:龙眼参多糖180mg·kg^-1。治疗结束后观察龙眼参多糖对SAMP8鼠Morris水迷宫逃避潜伏期,血清和脑组织SOD、GSH-Px、MDA、NO、Ach、AchE,脑组织NE、DA、5-HT、Glu、Gln、Asp、Asn、Gly、GABA、APP、Aβ、BACE、PS1、PS2、ApoE等指标的影响。以下是实验的主要结果:1、LYSP的提取、鉴定及分析结果LYSP经Sephadex-75凝胶柱层析,高效凝胶渗透色谱法（HPGPC）证实为高纯度多糖,紫外扫描显示无核酸和蛋白特征吸收峰,薄层色谱和气相色谱表明由葡萄糖和阿拉伯糖组成。2、LYSP对各种拟痴呆动物模型学习记忆的改善作用分别采用东莨菪碱、40%乙醇、D-半乳糖和快速老化小白鼠（senescenceaccelerated mouse prone/8,SAMP8）等建立各种小鼠学习记忆障碍模型,应用Y型水迷宫和Morris水迷宫观测各组小白鼠的学习记忆能力的变化。各种痴呆小鼠表现出明显的学习记忆障碍,龙眼参多糖能改善各种痴呆模型小白鼠的学习记忆能力。3、LYSP对NG108-15痴呆细胞模型的保护作用采用直接加药培养法和含药血清培养法观测龙眼参多糖对加入Aβ25-35的NG108-15细胞的生长状态、细胞增殖数、存活率、突起率及突起平均长度的影响。结果显示龙眼参多糖或其含药血清都能在一定程度上抑制Aβ25-35对细胞的损伤作用:细胞生长状态良好,细胞增殖数、存活率、突起率及突起平均长度均有明显提高。4、LYSP对SAMP8体内自由基代谢的影响采用分光光度法检测SAMP8鼠血清和脑组织SOD和GSH-Px活性、MDA和NO的含量。结果显示SAMP8组血清和脑组织SOD、GSH-Px活性明显降低,MDA和NO的含量明显升高,而龙眼参多糖能提高血清和脑组织SOD、GSH-Px活性,降低MDA和NO的含量明显,提示龙眼参多糖可能通过有效清除自由基而达到抗痴呆效果。5、LYSP对拟痴呆小鼠胆碱能神经递质的影响采用分光光度法检测D-半乳糖致拟痴呆动物模型和快速老化小白鼠模型血清和脑组织Ach含量和AchE活性。结果显示模型组、SAMP8空白组血清和脑组织AchE活性明显降低,Ach含量无明显变化。龙眼参多糖能降低血清和脑组织AchE活性,提示龙眼参多糖可能通过降低血清和脑组织AchE活性,减少Ach的降解而起抗痴呆作用。6、LYSP对SAMP8鼠脑组织单胺类神经递质的影响采用高效液相色谱法检测各组小鼠脑组织去甲肾上腺素（NE）、多巴胺（DA）、5-羟色胺（5-HT）含量。结果显示SAMP8空白组脑组织NE、DA、5-HT的含量明显降低,而龙眼参多糖能提高脑组织单胺类神经递质的含量,提示龙眼参多糖可有效调整单胺类神经递质的合成,具有良好的抗痴呆的作用。7、LYSP对SAMP8脑组织氨基酸类神经递质的影响采用高效液相色谱法检测各组小鼠脑组织氨基酸类神经递质含量。结果显示SAMP8空白组脑组织Glu、Gln、Asp、GABA含量明显升高,Asn、Gly有升高趋势,但无显著性;龙眼参多糖各剂量组能显著降低SAMP8鼠Glu、Asp含量,但对Gly的降低作用不明显。部分多糖剂量组对降低Gln、Asn和GABA含量作用明显。提示龙眼参多糖能显著降低SAMP8鼠脑组织Glu、Asp含量,纠正了SAMP8鼠脑区氨基酸类递质代谢的紊乱状态,使兴奋性和抑制性氨基酸神经递质最终趋向平衡。8、LYSP对SAMP8鼠脑组织APP、Aβ表达的影响采用免疫组化、图像分析测定SAMP8鼠脑内APP、Aβ平均光密度和阳性细胞数。结果显示龙眼参多糖各剂量能降低SAMP8脑内APP、Aβ平均光密度值和阳性细胞数,并且这种效应呈现一定的量效关系。提示龙眼参多糖能减少或抑制APP、Aβ的过度产生,保护神经元免受Aβ的神经毒性。9、LYSP对SAMP8鼠脑组织痴呆相关基因表达的影响采用半定量反转录聚合酶链反应（RT-PCR）测定SAMP8鼠脑内BACE、APP、PS1、PS2、ApoE mRNA含量。结果显示龙眼参多糖各剂量组能降低脑内BACE、APP、PS1、PS2 mRNA含量并呈现一定的量效关系,对ApoE mRNA含量的影响不明显。提示龙眼参多糖能下调SAMP8脑内BACE、APP、PS1、PS2基因的表达。综上所述,龙眼参多糖可能是通过清除自由基、减少Ach的降解、有效调整单胺类神经递质的合成和氨基酸类递质代谢的紊乱状态、抑制APP、Aβ的过度产生和抑制痴呆相关基因的表达等机制而达到抗痴呆作用。更多还原

【Abstract】 Alzheimer’s disease is one kind of disease characterized by recent memory handicap,senile plaque（SP）,neurofibrillary tangle（NFT）,and neuron loss in pathology and gradually neuron’s functional deterioration clinically,and it seriously affect the patient’s cognition function,memory function,language function and space cognition function,social activities ability,personal living ability and emotion personality etc.The cause and nosogenesis of disease are still not clear.Several theories now widely recognized are:neurotransmitter impairment theory;calcium homeostasis imbalance theory;free radicals injury theory;gene mutation theory;energy dysbolism theory;nerve cell apoptosis theory;excitatory transmitter toxicity;β-Amyloid deposition theory etc.LYS, the root of one plant named Millettia pulchra Kurz var laxior（Dunn）Z.Wei,is a commonly used Chinese medicinal herb in Guangxi province.It has the function of supplying QI and blood,increasing the human ability of immunity,anti-aging, anti-stress etc.It can also make the body strong and alleviate fatigue so as to be applied to treat poor memory in old people and children’s hypophrenia clinically. It can also be used for physical weak person and postnatal weakness women. Our research is to extract polysaccharides from LYS and to observe the effects of LYS polysaccharides on anti-dementia and its mechanism.In these experiments,NG108-15 cells were used to establish cell model of Alzheimer’s disease induced by Aβ25-35.Kunming mice were used to establish learning and memory disorder model induced by scopomamine,40%ethanol, D-galactose respectively.Senescence accelerated mouse（SAM）was used to establish the AD model as well.The subgroup and administration of Kunming mice are:（1）normal control group:normal sodium,30ml·kg^-1;（2）model group: normal sodium,30ml·kg^-1;（3）pisacetam control group:pisacetam,0.5g·kg^-1;（4） low-dose of LYSP group:LYSP,45mg·kg^-1;（5）mid-dose of LYSP group: 90mg·kg^-1;（6）high-dose of LYSP group:LYSP,180mg·kg^-1.After treatment, the effects of LYSP on correct frequency in Y-water maze test,Ach and AchE in mice serum or brain were detected.The subgroup and administration of SAM are:（1）SAMR1 group:normal sodium,30ml·kg^-1;（2）SAMP8 untreated control group:normal sodium,30ml·kg^-1;（3）huperzine A control group:huperzine A, 0.02mg·kg^-1;（4）low-dose of LYSP group:LYSP,45mg·kg^-1;（5）mid-dose of LYSP group:90mg·kg^-1;（6）high-dose of LYSP group:LYSP,180mg·kg^-1.After treatment,the effects of LYSP on escape latency in Morris water maze test, SOD,GSH-Px,MDA,NO,Ach and AchE in mice serum or brain,NE,DA, 5-HT,Glu,Gin,Asp,Asn,Gly,GABA,APP,Aβ,BACE,PS1,PS2 and ApoE in mice brain were observed.The main results are as follows:1.The extraction,identification and analysis of LYSP compositionLYSP didn’t contain protein and nucleic acid.It showed high purity of polysaccharides when identified by means of Sephadex G-75 column or high performance gel permeation chromatography（HPGPC）.It was composed of glucose and arabinose.2.Improvement of LYSP on the learning and memory abilities in learning and memory disorder modelScopomamine,40%alcohol,D-galactose and senescence accelerated mouse prone/8 were used to establish learning and memory disorder model respectively.Then Y-water maze and Morris water maze test were used to assess the change of learning and memory abilities of each group.The results showed that all kinds of dementia model were significant impaired in learning and memory abilities,and LYSP-treated group could significantly improve the learning and memory abilities.3.Protective effect of LYSP on NG108-15 dementia cells modelLYSP and the serum containing LYSP were used to culture NG108-15 cell model of Alzheimer induced by Aβ25-35.The cell morphology,proliferation, survival rate,neurite outgrowth and average length were observed.The results showed that LYSP or the serum containing LYSP could protect NG108-15 cells from Aβ25-35 neurotoxicity:the cells morphology was at good condition,and the cell viability,proliferation,survival rate,neurite outgrowth and average length were significantly elevated.4.Effects of LYSP on free radical metabolism in SAMP8Spectrophotometry was used to detect SOD and GSH-Px activities,MDA and NO contents in SAMP8 serum and brain.The results showed that SOD and GSH-Px activities were decreased significantly,and MDA and NO contents were increased significantly in model group or SAMP8 serum and brain. LYSP-treated group could enhence SOD and GSH-Px activities and decrease significantly MDA and NO contents in SAMP8 serum and brain.It suggested that LYSP could scavenge effectively free radical for anti-dementia goal.5.Effects of LYSP on cholinergic neurotransmitter in dememntia modelSpectrophotometry was used to detect Ach content and AchE activity in dementia animal model induced by D-galactose and SAMP8 model serum and brain.The results showed that AchE activity decreased significantly and Ach content had no significant difference in SAMP8 serum and brain.LYSP-treated group could decrease AchE activity.It suggested that LYSP could reduce the degradation of Ach by decreaseing AchE activity for anti-dementia goal. 6.Effects of LYSP on monoamine neurotransmitter in SAMP8 brainThe NE,DA and 5-HT contents in brain were detected with HPLC.The results showed that the NE,DA and 5-HT contents in SAMP8 brain were decreased significantly and LYSP-treated group could increase the NE,DA and 5-HT contents.It suggested that LYSP has obvious anti-dementia effects,which could regulate effectively the disorderly metabolized monoamine transmitter.7.Effects of LYSP on amino acids neurotransmitter in SAMP8 brainThe contents of amino acids neurotransmitter in brain were detected with HPLC.The results showed that the contents of GLU,GLN,ASP and GABA of SAMP8 group increased significantly in brain,and the contents of ASN and GLY were not significant increased.In LYSP-treated group,the contents of GLU and ASP decreased significantly,but the content of GLY was not obvious changed.The contents of GLN,ASN and GABA were decreased by treatment with LYSP.It suggested that LYSP could obviously decrease the contents of GLU and ASP,retrieve the metabolic disorder of amino acid neurotransmitter in SAMP8 brain,and make the balance of the excitatory and inhibitory amino acid neurotransmitter.8.Effect of LYSP on expressions of APP and AB in SAMP8 brainAPP and Aβaverage optical density and their immunoreactive neurons in brain tissue were determined by immunohistochemical analysis and computer image analysis.The results showed that LYSP-treated groups could decrease significantly APP and Aβaverage optical density and their immunoreactive neurons in brain tissue of SAMP8.It showed a dose-effect relationship in some degrees.It suggested that LYSP could reduce or inhibit the over-production of APP and Aβin the brain,so as to protect the neurons from the neurontoxicity ofβ-Amyloid. 9.Effect of LYSP on dementia-related gene expressions in SAMP8 brainThe mRNA contents of BACE,APP,PS1,PS2 and ApoE in brain tissue were assayed by reverse transcription polymerase chain reaction.The results showed that the LYSP-treated groups could reduce the mRNA contents of BACE,APP,PS1 and PS2,which showed a dose-effect relationship in some degrees.But it could not affect the content of ApoE.It suggested that LYSP could inhibit the expressions of BACE,APP,PS1,and PS2 in SAMP8 brain.In summary,the present results suggest that LYSP is effective in anti-dementia.As a possible mechanism,LYSP can scavenge free radicals, reduce the degradation of Ach,regulate effectively the disorderly metabolized monoamine and amino acid neurotransmitter,inhibit the over-production of APP and Aβ,and inhibit the expressions of dementia-related gene.更多还原