本文關鍵詞：水溶性人參寡糖改善東莨菪堿誘導的學習障礙及相關機制研究，由筆耕文化傳播整理發(fā)布。

        學習與記憶是大腦的重要神經活動，是人類正常生活所必需具備的能力。大腦海馬區(qū)與學習、記憶、認知功能關系最為密切，海馬結構或功能發(fā)生變化都會導致認知障礙。老年癡呆癥就是一種漸進的神經變性性腦疾病，其病理學特征主要是海馬和基底前腦神經細胞損失，導致記憶能力衰退。對老年癡呆的發(fā)病機制涉及多種學說，我們的研究涉及：①“膽堿能學說”——老年癡呆癥患者腦內的膽堿能神經元減少，導致乙酰膽堿(acetylcholine, ACh)合成、儲存和釋放減少，引發(fā)認知功能障礙。②“氧自由基學說”——老年癡呆癥患者的腦內氧自由基增多，導致脂質過氧化，蛋白質和細胞功能損傷，引發(fā)神經元退化、變性和凋亡。ACh是參與學習記憶的重要神經遞質。中樞膽堿能神經末梢釋放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，記憶功能的強弱主要取決于腦內ACh的含量，當腦中ACh含量高時，學習記憶能力增強，而當膽堿能系統(tǒng)受到損傷或異常導致ACh含量低時，學習記憶能力減弱或喪失。老年癡呆癥患者，腦內的ACh含量明顯減少，進而引起記憶和識別功能障礙等。因此，臨床上用乙酰膽堿酯酶抑制劑增加腦內ACh濃度來治療老年癡呆癥取得了一定的療效。氧化應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氧化應激的發(fā)生。過氧化氫酶（catalase，CAT）是體內廣泛存在的催化過氧化氫分解的酶，是體內重要的自由基清除劑，，對清除自由基和防止脂質過氧化發(fā)揮重要作用，能夠防止神經元退化、變性、凋亡、延緩神經元的衰老，維持學習記憶能力。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、保護和修復神經元，改善腦細胞功能，起到抗衰老和改善學習記憶的作用。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。過去十年期間，人們更多關注人參皂苷和人參多糖的結構及生物活性，對于人參寡糖的研究則非常有限。人參寡糖分子結構簡單且分子量小，易于腸道的消化吸收，體內利用率較高。研究發(fā)現(xiàn)，人參寡糖能顯著增強免疫系統(tǒng)的功能。另外，寡糖對腦內ACh缺乏導致的認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）誘導的學習記憶缺欠小鼠注射水溶性人參寡糖（water-soluble ginseng oligosaccharides，WGOS），通過行為觀測和相關生化物質檢測觀察WGOS的療效。我們的研究顯示：1﹑在Morris水迷宮的訓練期，單獨注射生理鹽水和兩個劑量的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，兩者間無顯著性差異（P﹥0.05），表明WGOS不改變正常小鼠的學習獲得情況。Scop組小鼠的平均逃逸潛伏期幾乎不變，表明注射Scop后的小鼠學習能力顯著下降。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誘導的學習獲得障礙。2﹑在Morris水迷宮的定位航行測試中，單獨注射鹽水與WGOS40、WGOS80的小鼠，其平均逃逸潛伏期﹑總路程無顯著性差異（P﹥0.05），表明單獨注射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誘導的記憶障礙。3﹑在Morris水迷宮的空間搜索測試中，我們測定了穿過平臺次數(shù)、經過有效區(qū)次數(shù)、總路程等指標。結果發(fā)現(xiàn)，單獨注射鹽水與WGOS40、WGOS80的小鼠間無顯著性差異（P﹥0.05），表明單獨注射WGOS不改變正常小鼠的空間記憶情況。Scop組小鼠上述指標均明顯高于鹽水對照組（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組小鼠上述指標接近鹽水對照組（P﹥0.05）但明顯低于Scop組（P﹤0.01），表明WGOS能顯著改善由Scop誘導的空間記憶障礙。4﹑行為測試后，我們測定了小鼠海馬組織的ACh含量。結果表明:單獨注射鹽水與WGOS40、WGOS80的小鼠海馬中ACh含量無顯著性差異（P﹥0.05），表明單獨注射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誘導的海馬ACh含量降低的狀況。5﹑我們也測定了小鼠海馬中AChE活性。結果表明：單獨注射Saline與WGOS40、WGOS80的小鼠海馬中AChE活性無顯著性差異（P﹥0.05），表明單獨注射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誘導的AChE活性升高的狀況。6﹑我們還測定了小鼠海馬中CAT活性。結果表明：單獨注射Saline與WGOS40、WGOS80的小鼠海馬中CAT活性無顯著性差異（P﹥0.05），表明單獨注射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誘導的CAT活性降低的狀況。由此，我們得出如下結論：WGOS能明顯改善Scop誘導的空間學習記憶缺陷，但不影響正常小鼠的學習記憶過程。其機制涉及：①WGOS能夠改善由Scop誘導的小鼠海馬組織中AChE活性升高，進而恢復ACh含量，改善空間學習記憶能力。②W GOS能顯著改善由Scop誘導的小鼠海馬組織中CAT活性降低，減少自由基產生，減輕腦內氧化應激，防止神經細胞功能損傷，改善學習記憶能力。

    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的學習障礙及相關機制研究

前言4-6中文摘要6-13Abstract13-23第1章 文獻綜述29-45    1.1 學習記憶的生理機制及認知障礙29-39        1.1.1 膽堿能系統(tǒng)參與學習記憶過程30-33        1.1.2 氧化性應激也被涉及老年癡呆癥的發(fā)病過程33-35        1.1.3 海馬是學習記憶的重要核團35-38        1.1.4 評價實驗動物學習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 實驗材料與方法45-55    2.1 實驗材料45-46        2.1.1 主要試劑45        2.1.2 主要儀器設備45-46        2.1.3 實驗動物46    2.2 實驗方法46-51        2.2.1 實驗動物藥物處理46-47        2.2.2 Morris 水迷宮測試47-50        2.2.3 組織樣品的收集50-51    2.3 生化檢測51-54        2.3.1 乙酰膽堿 ACh 含量測定51-52        2.3.2 乙酰膽堿酯酶 AChE 活性測定52-53        2.3.3 過氧化氫酶 CAT 活性測定53        2.3.4 總蛋白濃度的測定53-54    2.4 數(shù)據(jù)處理54-55第3章 實驗結果55-64    3.1 連續(xù)應用 WGOS 的小鼠體重明顯增加55-56    3.2 Scop 誘導的空間學習記憶障礙小鼠模型的制備56    3.3 WGOS 改善 Scop 誘導的空間學習記憶障礙的行為學分析56-61        3.3.1 WGOS 對 Scop 誘導的學習障礙小鼠水迷宮訓練的影響56-57        3.3.2 WGOS 對 Scop 誘導的學習障礙小鼠水迷宮定位航行測試的影響57-59        3.3.3 WGOS 對 Scop 誘導的學習障礙小鼠水迷宮空間搜索測試的影響59-61    3.4 WGOS 對 Scop 誘導的學習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 誘導的學習障礙小鼠海馬中 ACh 含量61-62        3.4.2 WGOS 改善 Scop 誘導的學習障礙小鼠海馬中 AChE 活性62-63    3.5 WGOS 改善 Scop 誘導的學習障礙小鼠海馬中 CAT 活性63-64第4章 討論64-73第5章 結論73-74參考文獻74-89作者簡介及在學期間所取得的科研成果89-90致謝90-91

  本文關鍵詞：水溶性人參寡糖改善東莨菪堿誘導的學習障礙及相關機制研究，由筆耕文化傳播整理發(fā)布。