本文關(guān)鍵詞：水溶性人參寡糖改善東莨菪堿誘導(dǎo)的學(xué)習(xí)障礙及相關(guān)機(jī)制研究，由筆耕文化傳播整理發(fā)布。

        學(xué)習(xí)與記憶是大腦的重要神經(jīng)活動(dòng)，是人類正常生活所必需具備的能力。大腦海馬區(qū)與學(xué)習(xí)、記憶、認(rèn)知功能關(guān)系最為密切，海馬結(jié)構(gòu)或功能發(fā)生變化都會(huì)導(dǎo)致認(rèn)知障礙。老年癡呆癥就是一種漸進(jìn)的神經(jīng)變性性腦疾病，其病理學(xué)特征主要是海馬和基底前腦神經(jīng)細(xì)胞損失，導(dǎo)致記憶能力衰退。對(duì)老年癡呆的發(fā)病機(jī)制涉及多種學(xué)說(shuō)，我們的研究涉及：①“膽堿能學(xué)說(shuō)”——老年癡呆癥患者腦內(nèi)的膽堿能神經(jīng)元減少，導(dǎo)致乙酰膽堿(acetylcholine, ACh)合成、儲(chǔ)存和釋放減少，引發(fā)認(rèn)知功能障礙。②“氧自由基學(xué)說(shuō)”——老年癡呆癥患者的腦內(nèi)氧自由基增多，導(dǎo)致脂質(zhì)過(guò)氧化，蛋白質(zhì)和細(xì)胞功能損傷，引發(fā)神經(jīng)元退化、變性和凋亡。ACh是參與學(xué)習(xí)記憶的重要神經(jīng)遞質(zhì)。中樞膽堿能神經(jīng)末梢釋放ACh，作用于煙堿受體或毒蕈堿受體而發(fā)揮生理作用。ACh作用的終止依靠突觸間隙中乙酰膽堿酯酶（acetylcholinesterase，AChE）的作用。研究表明，記憶功能的強(qiáng)弱主要取決于腦內(nèi)ACh的含量，當(dāng)腦中ACh含量高時(shí)，學(xué)習(xí)記憶能力增強(qiáng)，而當(dāng)膽堿能系統(tǒng)受到損傷或異常導(dǎo)致ACh含量低時(shí)，學(xué)習(xí)記憶能力減弱或喪失。老年癡呆癥患者，腦內(nèi)的ACh含量明顯減少，進(jìn)而引起記憶和識(shí)別功能障礙等。因此，臨床上用乙酰膽堿酯酶抑制劑增加腦內(nèi)ACh濃度來(lái)治療老年癡呆癥取得了一定的療效。氧化應(yīng)激也是老年癡呆的影響因素之一，因此一些抗氧化劑被研究用于減少患者腦內(nèi)氧化應(yīng)激的發(fā)生。過(guò)氧化氫酶（catalase，CAT）是體內(nèi)廣泛存在的催化過(guò)氧化氫分解的酶，是體內(nèi)重要的自由基清除劑，，對(duì)清除自由基和防止脂質(zhì)過(guò)氧化發(fā)揮重要作用，能夠防止神經(jīng)元退化、變性、凋亡、延緩神經(jīng)元的衰老，維持學(xué)習(xí)記憶能力。臨床上輔助治療老年癡呆癥的抗氧化劑主要有維生素E、褪黑激素、單胺氧化酶B抑制劑等，它們具有激活、保護(hù)和修復(fù)神經(jīng)元，改善腦細(xì)胞功能，起到抗衰老和改善學(xué)習(xí)記憶的作用。人參為五加科多年生草本植物的根，它能夠治療許多年齡相關(guān)疾病，包括抗高血壓和記憶缺失等。人參含有多種類型的化學(xué)活性成分，如皂苷類、多糖、寡糖、多肽、脂肪酸、氨基酸、揮發(fā)油等。過(guò)去十年期間，人們更多關(guān)注人參皂苷和人參多糖的結(jié)構(gòu)及生物活性，對(duì)于人參寡糖的研究則非常有限。人參寡糖分子結(jié)構(gòu)簡(jiǎn)單且分子量小，易于腸道的消化吸收，體內(nèi)利用率較高。研究發(fā)現(xiàn)，人參寡糖能顯著增強(qiáng)免疫系統(tǒng)的功能。另外，寡糖對(duì)腦內(nèi)ACh缺乏導(dǎo)致的認(rèn)知障礙有一定治療效果。基于以上研究，我們給東莨菪堿（Scopolamine，Scop）誘導(dǎo)的學(xué)習(xí)記憶缺欠小鼠注射水溶性人參寡糖（water-soluble ginseng oligosaccharides，WGOS），通過(guò)行為觀測(cè)和相關(guān)生化物質(zhì)檢測(cè)觀察WGOS的療效。我們的研究顯示：1﹑在Morris水迷宮的訓(xùn)練期，單獨(dú)注射生理鹽水和兩個(gè)劑量的WGOS（40mg/kg，80mg/kg，記作WGOS40和WGOS80），小鼠的平均逃逸潛伏期是逐漸縮短的，兩者間無(wú)顯著性差異（P﹥0.05），表明WGOS不改變正常小鼠的學(xué)習(xí)獲得情況。Scop組小鼠的平均逃逸潛伏期幾乎不變，表明注射Scop后的小鼠學(xué)習(xí)能力顯著下降。WGOS40+Scop組和WGOS80+Scop組小鼠平均逃逸潛伏期接近鹽水對(duì)照組（P﹥0.05）但明顯低于Scop組（P﹤0.01），表明WGOS能顯著改善由Scop誘導(dǎo)的學(xué)習(xí)獲得障礙。2﹑在Morris水迷宮的定位航行測(cè)試中，單獨(dú)注射鹽水與WGOS40、WGOS80的小鼠，其平均逃逸潛伏期﹑總路程無(wú)顯著性差異（P﹥0.05），表明單獨(dú)注射WGOS不改變正常小鼠的記憶情況。Scop組小鼠的平均逃逸潛伏期﹑總路程明顯高于鹽水對(duì)照組（P＜0.01），表明注射Scop后的小鼠記憶能力顯著下降。WGOS40+Scop組和WGOS80+Scop小鼠平均逃逸潛伏期﹑總路程接近鹽水對(duì)照組（P﹥0.05）但明顯低于Scop組（P﹤0.01），表明WGOS能顯著改善由Scop誘導(dǎo)的記憶障礙。3﹑在Morris水迷宮的空間搜索測(cè)試中，我們測(cè)定了穿過(guò)平臺(tái)次數(shù)、經(jīng)過(guò)有效區(qū)次數(shù)、總路程等指標(biāo)。結(jié)果發(fā)現(xiàn)，單獨(dú)注射鹽水與WGOS40、WGOS80的小鼠間無(wú)顯著性差異（P﹥0.05），表明單獨(dú)注射WGOS不改變正常小鼠的空間記憶情況。Scop組小鼠上述指標(biāo)均明顯高于鹽水對(duì)照組（P＜0.01），表明注射Scop后的小鼠空間記憶能力顯著下降。WGOS40+Scop組和WGOS80+Scop組小鼠上述指標(biāo)接近鹽水對(duì)照組（P﹥0.05）但明顯低于Scop組（P﹤0.01），表明WGOS能顯著改善由Scop誘導(dǎo)的空間記憶障礙。4﹑行為測(cè)試后，我們測(cè)定了小鼠海馬組織的ACh含量。結(jié)果表明:單獨(dú)注射鹽水與WGOS40、WGOS80的小鼠海馬中ACh含量無(wú)顯著性差異（P﹥0.05），表明單獨(dú)注射WGOS不改變正常小鼠海馬中ACh含量。Scop組小鼠海馬中ACh含量低于鹽水對(duì)照組（P﹤0.05），而WGOS40+Scop組和WGOS80+Scop組小鼠海馬中ACh含量接近鹽水對(duì)照組（P﹥0.05）但明顯高于Scop組（P﹤0.05），表明WGOS能顯著改善由Scop誘導(dǎo)的海馬ACh含量降低的狀況。5﹑我們也測(cè)定了小鼠海馬中AChE活性。結(jié)果表明：?jiǎn)为?dú)注射Saline與WGOS40、WGOS80的小鼠海馬中AChE活性無(wú)顯著性差異（P﹥0.05），表明單獨(dú)注射WGOS不改變正常小鼠海馬中AChE活性。Scop組小鼠海馬中AChE活性明顯高于鹽水對(duì)照組（P﹤0.05）。WGOS40+Scop組和WGOS80+Scop組小鼠海馬中AChE活性接近鹽水對(duì)照組（P﹥0.05）但明顯低于Scop組（P﹤0.05），表明WGOS能顯著改善由Scop誘導(dǎo)的AChE活性升高的狀況。6﹑我們還測(cè)定了小鼠海馬中CAT活性。結(jié)果表明：?jiǎn)为?dú)注射Saline與WGOS40、WGOS80的小鼠海馬中CAT活性無(wú)顯著性差異（P﹥0.05），表明單獨(dú)注射WGOS不改變正常小鼠海馬中CAT活性。Scop組小鼠海馬中CAT活性明顯低于鹽水對(duì)照組（P﹤0.01），WGOS40+Scop組和WGOS80+Scop組小鼠海馬中CAT活性接近鹽水對(duì)照組（P﹥0.05）但明顯高于Scop組（P﹤0.05），表明WGOS能顯著改善由Scop誘導(dǎo)的CAT活性降低的狀況。由此，我們得出如下結(jié)論：WGOS能明顯改善Scop誘導(dǎo)的空間學(xué)習(xí)記憶缺陷，但不影響正常小鼠的學(xué)習(xí)記憶過(guò)程。其機(jī)制涉及：①WGOS能夠改善由Scop誘導(dǎo)的小鼠海馬組織中AChE活性升高，進(jìn)而恢復(fù)ACh含量，改善空間學(xué)習(xí)記憶能力。②W GOS能顯著改善由Scop誘導(dǎo)的小鼠海馬組織中CAT活性降低，減少自由基產(chǎn)生，減輕腦內(nèi)氧化應(yīng)激，防止神經(jīng)細(xì)胞功能損傷，改善學(xué)習(xí)記憶能力。

    Learning and memory is an important function of the brain and anecessary feature for human life. The hippocampus is closely related tolearning, memory and cognitive function. Structural or functional changesin the hippocampus would lead to cognitive impairment. Alzheimer’sdisease (AD) is a progressive neurodegenerative brain disorder, and itsneuropathological feature is the loss of nerve cell in the hippocampus andbasal forebrain, resulting in degeneration of the memory capacity.Multiple theories such as the cholinergic theory and the oxygen freeradical theory have been involved in the pathogenesis of AD. Thecholinergic theory, which is characterized by deficiencies of acetylcholine (ACh), has been considered the impairment of cholinergic neurons andneuronal dysfunction in the brain of Alzheimer’s disease patients. Theoxidation free radical theory, which is characterized by increase ofoxyradical, has been considered induction of lipid peroxidation andcausing neuronal degeneration, denaturation and apoptosis in Alzheimer’sbrain.ACh is an important neurotransmitter involved in learning andmemory. The cholinergic nerve endings can release ACh and ACh exertsphysiological effect through two receptors, nicotinic receptors andmuscarinic receptors, in central nervous system. The termination of ACheffect depends on acetylcholinesterase (AChE), which can decomposeACh, in the cholinergic synaptic cleft. Some Researches have showedthat memory function principally depends on ACh content in brain and isattenuated when ACh is lower level. In the brain of AD patients, ACh content was significantly reduced, causing memory and cognitivedysfunction. Therefore, AChE inhibitors have been used to treat cognitiveimpairment in AD patients.Oxidative stress is also one of the main affecting factors in AD, soseveral antioxidants have been studied for the reduction of oxidativestress occurring in the brain of the patients. Catalase (CAT), which is anenzyme catalyzed decomposition of hydrogen peroxide, is an importantfree radical scavenger in vivo and plays an important role in preventinglipid peroxidation. Therefore, CAT could maintain learning and memoryabilities due to preventing neuronal degeneration and apoptosis anddelaying neurons senescence. Several antioxidants such as vitamin E,melatonin and monoamine oxidase B inhibitors have been tested for theadjuvant treatment of AD in therapeutics. They could activate, protect andrepair the neurons, and could improve the function of brain cells, so they play a role in anti-aging and amelioration learning and memory.Ginseng is root of Araliaceae perennial herb and its infusions orextracts are reported to be effective for a large number of medicalproblems associated with aging, including essential hypertension andmemory loss. Ginseng contains multifold chemical active componentswhich including saponins, polysaccharides, oligosaccharides,polypeptides, fatty acids, amino acids and aetherolea. During the pastdecades, numerous researchers have focused on structures andbioactivities of ginsenosides and ginseng polysaccharides. As for thevaluable oligosaccharide in ginseng, the reports are quite limited.The digestion and absorption of ginseng oligosaccharides iseffortless in intestinal tract and its utilization rate is higher in vivo,because it has simple molecular structure and small molecular weight.Some researches found that ginseng oligosaccharides can significantly enhance the function of immune system. Recently, it has been reportedthat oligosaccharides have a significant effect in treatment of cognitiveimpairment caused by deficiency of ACh. Based on these studies, weinjected intraperitoneally water-soluble ginseng oligosaccharides(designated as WGOS), which were obtained form warm-water extract ofPanax ginseng roots, in memory impairment mice induced byscopolamine (Scop) to detect therapeutic effect of WGOS throughbehavioral and biochemical experiments. Our research showed:1. During the training days of Morris water maze, the mean escapelatency was gradually shortened in both the saline alone and WGOS40,WGOS80alone mice and no significant difference among the treatmentgroups (P＞0.05), indicating that the WGOS could not influence spatiallearning capacity in normal mice. The mean escape latency was almostconstant in Scop alone group during training days, demonstrating that spatial learning capacity was decreased after Scop administration.However, WGOS40+Scop and WGOS80+Scop group showed that themean escape latency approach to saline control group (P﹥0.05) and itwere significantly shorter than the Scop alone group (P﹤0.01),suggesting that WGOS could improve Scop-induced spatial learningdeficits.2. During the test of place navigation in Morris water maze, themean escape latency and total distance were no significant differenceamong saline alone and WGOS40, WGOS80alone mice (P﹥0.05),indicating that WGOS could not influence memory capacity in normalmice. The mean escape latency and total distance were significantlyhigher in Scop alone group than the saline group(P﹤0.01), demonstratingdecreased memory capacity after Scop administration. However,WGOS40+Scop and WGOS80+Scop group showed that the mean escape latency and total distance approach to the saline control group (P﹥0.05) and were significantly lower than the Scop alone group (P﹤0.01),suggesting that WGOS could improve Scop-induced memory deficits.3. During the test of spatial search in Morris water maze, we testedfour indexes, the frequency traversed the platform and active area, totaldistance in each treatment group. They were no significant differenceamong saline alone and WGOS40, WGOS80alone mice (P>0.05),indicating that WGOS could not influence spatial memory capacity innormal mice. They was significantly higher in Scop alone group than thesaline control (P <0.01), demonstrating decreased spatial memorycapacity after Scop administration. However, WGOS40+Scop andWGOS80+Scop group showed that those indexes approach to the salinecontrol group (P﹥0.05) and were significantly lower (P﹤0.01) than theScop alone group, suggesting WGOS could improve Scop-induced spatial memory deficits.4. After behavioral tests, we measured the ACh content in micehippocampus of each treatment group. Our result indicated that AChcontent in hippocampus was no significant difference among saline aloneand WGOS40, WGOS80alone mice (P>0.05), indicating that WGOScould not influence the ACh content in hippocampus of normal mice. TheACh content was significantly lower in hippocampus of Scop alone micethan the saline control group (P <0.05). However, WGOS40+Scop andWGOS80+Scop group showed that the ACh content in hippocampusapproach to the saline control group (P>0.05), and was significantlyhigher than the Scop alone group (P <0.05), suggesting WGOS couldimprove Scop-induced the ACh content in hippocampus.5. We also measured the AChE activity in hippocampus of eachtreament group. Our result indicated that the AChE activity in hippocampus was no significant difference among saline alone andWGOS40, WGOS80alone mice (P>0.05), indicating that WGOS couldnot influence the AChE activity in hippocampus of normal mice. TheAChE activity in hippocampus was significantly higher in Scop alonegroup than the saline control group (P <0.05). However, WGOS40+Scopand WGOS80+Scop group showed that the AChE activity inhippocampus approach to the saline control group (P>0.05) and wassignificantly lower than the Scop alone group (P <0.05), suggesting thatWGOS could improve Scop-induced increasing of the AChE activity inhippocampus.6. We also measured the CAT activity in hippocampus of each treamentgroup. Our result indicated that the CAT activity in hippocampus was nosignificant difference among saline alone and WGOS40, WGOS80alonemice (P>0.05), indicating that WGOS could not influence the CAT activity in hippocampus of normal mice. The CAT activity inhippocampus was significantly lower in Scop alone group than the salinecontrol group (P <0.01). However, WGOS40+Scop and WGOS80+Scop group showed that the CAT activity in hippocampus approach to thesaline control group (P>0.05), and was significantly higher than the Scopalone group (P <0.01), suggesting that WGOS could improveScop-induced reduction of the CAT activity in hippocampus.In conclusions, the present study demonstrates that WGOS couldsignificantly improvement Scop-induced spatial learning and memorydefects, but could not influence learning and memory in normal mice.WGOS had therapeutic efficacy for Scop-induced learning and memorydefects by concurrently acting on multiple mechanisms, includingdecreasing ACh content, inhibiting AChE activity and augmenting CATactivity in hippocampus. Therefore, WGOS may be a potential drug for the treatment of AD.

水溶性人參寡糖改善東莨菪堿誘導(dǎo)的學(xué)習(xí)障礙及相關(guān)機(jī)制研究

前言4-6中文摘要6-13Abstract13-23第1章 文獻(xiàn)綜述29-45    1.1 學(xué)習(xí)記憶的生理機(jī)制及認(rèn)知障礙29-39        1.1.1 膽堿能系統(tǒng)參與學(xué)習(xí)記憶過(guò)程30-33        1.1.2 氧化性應(yīng)激也被涉及老年癡呆癥的發(fā)病過(guò)程33-35        1.1.3 海馬是學(xué)習(xí)記憶的重要核團(tuán)35-38        1.1.4 評(píng)價(jià)實(shí)驗(yàn)動(dòng)物學(xué)習(xí)記憶功能的常用方法38-39    1.2 人參及其生物活性成分的藥理性作用39-45第2章 實(shí)驗(yàn)材料與方法45-55    2.1 實(shí)驗(yàn)材料45-46        2.1.1 主要試劑45        2.1.2 主要儀器設(shè)備45-46        2.1.3 實(shí)驗(yàn)動(dòng)物46    2.2 實(shí)驗(yàn)方法46-51        2.2.1 實(shí)驗(yàn)動(dòng)物藥物處理46-47        2.2.2 Morris 水迷宮測(cè)試47-50        2.2.3 組織樣品的收集50-51    2.3 生化檢測(cè)51-54        2.3.1 乙酰膽堿 ACh 含量測(cè)定51-52        2.3.2 乙酰膽堿酯酶 AChE 活性測(cè)定52-53        2.3.3 過(guò)氧化氫酶 CAT 活性測(cè)定53        2.3.4 總蛋白濃度的測(cè)定53-54    2.4 數(shù)據(jù)處理54-55第3章 實(shí)驗(yàn)結(jié)果55-64    3.1 連續(xù)應(yīng)用 WGOS 的小鼠體重明顯增加55-56    3.2 Scop 誘導(dǎo)的空間學(xué)習(xí)記憶障礙小鼠模型的制備56    3.3 WGOS 改善 Scop 誘導(dǎo)的空間學(xué)習(xí)記憶障礙的行為學(xué)分析56-61        3.3.1 WGOS 對(duì) Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠水迷宮訓(xùn)練的影響56-57        3.3.2 WGOS 對(duì) Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠水迷宮定位航行測(cè)試的影響57-59        3.3.3 WGOS 對(duì) Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠水迷宮空間搜索測(cè)試的影響59-61    3.4 WGOS 對(duì) Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠海馬中膽堿能系統(tǒng)的影響61-63        3.4.1 WGOS 改善 Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠海馬中 ACh 含量61-62        3.4.2 WGOS 改善 Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠海馬中 AChE 活性62-63    3.5 WGOS 改善 Scop 誘導(dǎo)的學(xué)習(xí)障礙小鼠海馬中 CAT 活性63-64第4章 討論64-73第5章 結(jié)論73-74參考文獻(xiàn)74-89作者簡(jiǎn)介及在學(xué)期間所取得的科研成果89-90致謝90-91

  本文關(guān)鍵詞：水溶性人參寡糖改善東莨菪堿誘導(dǎo)的學(xué)習(xí)障礙及相關(guān)機(jī)制研究，由筆耕文化傳播整理發(fā)布。