水溶性人参寡糖改善东莨菪碱诱导的学习障碍及相关机制研究

  本文关键词：水溶性人参寡糖改善东莨菪碱诱导的学习障碍及相关机制研究，由笔耕文化传播整理发布。

        学习与记忆是大脑的重要神经活动，是人类正常生活所必需具备的能力。大脑海马区与学习、记忆、认知功能关系最为密切，海马结构或功能发生变化都会导致认知障碍。老年痴呆症就是一种渐进的神经变性性脑疾病，其病理学特征主要是海马和基底前脑神经细胞损失，导致记忆能力衰退。对老年痴呆的发病机制涉及多种学说，我们的研究涉及：①“胆碱能学说”——老年痴呆症患者脑内的胆碱能神经元减少，导致乙酰胆碱(acetylcholine, ACh)合成、储存和释放减少，引发认知功能障碍。②“氧自由基学说”——老年痴呆症患者的脑内氧自由基增多，导致脂质过氧化，蛋白质和细胞功能损伤，引发神经元退化、变性和凋亡。ACh是参与学习记忆的重要神经递质。中枢胆碱能神经末梢释放ACh，作用于烟碱受体或毒蕈碱受体而发挥生理作用。ACh作用的终止依靠突触间隙中乙酰胆碱酯酶（acetylcholinesterase，AChE）的作用。研究表明，记忆功能的强弱主要取决于脑内ACh的含量，当脑中ACh含量高时，学习记忆能力增强，而当胆碱能系统受到损伤或异常导致ACh含量低时，学习记忆能力减弱或丧失。老年痴呆症患者，脑内的ACh含量明显减少，进而引起记忆和识别功能障碍等。因此，临床上用乙酰胆碱酯酶抑制剂增加脑内ACh浓度来治疗老年痴呆症取得了一定的疗效。氧化应激也是老年痴呆的影响因素之一，因此一些抗氧化剂被研究用于减少患者脑内氧化应激的发生。过氧化氢酶（catalase，CAT）是体内广泛存在的催化过氧化氢分解的酶，是体内重要的自由基清除剂，，对清除自由基和防止脂质过氧化发挥重要作用，能够防止神经元退化、变性、凋亡、延缓神经元的衰老，维持学习记忆能力。临床上辅助治疗老年痴呆症的抗氧化剂主要有维生素E、褪黑激素、单胺氧化酶B抑制剂等，它们具有激活、保护和修复神经元，改善脑细胞功能，起到抗衰老和改善学习记忆的作用。人参为五加科多年生草本植物的根，它能够治疗许多年龄相关疾病，包括抗高血压和记忆缺失等。人参含有多种类型的化学活性成分，如皂苷类、多糖、寡糖、多肽、脂肪酸、氨基酸、挥发油等。过去十年期间，人们更多关注人参皂苷和人参多糖的结构及生物活性，对于人参寡糖的研究则非常有限。人参寡糖分子结构简单且分子量小，易于肠道的消化吸收，体内利用率较高。研究发现，人参寡糖能显著增强免疫系统的功能。另外，寡糖对脑内ACh缺乏导致的认知障碍有一定治疗效果。基于以上研究，我们给东莨菪碱（Scopolamine，Scop）诱导的学习记忆缺欠小鼠注射水溶性人参寡糖（water-soluble ginseng oligosaccharides，WGOS），通过行为观测和相关生化物质检测观察WGOS的疗效。我们的研究显示：1﹑在Morris水迷宫的训练期，单独注射生理盐水和两个剂量的WGOS（40mg/kg，80mg/kg，记作WGOS40和WGOS80），小鼠的平均逃逸潜伏期是逐渐缩短的，两者间无显著性差异（P﹥0.05），表明WGOS不改变正常小鼠的学习获得情况。Scop组小鼠的平均逃逸潜伏期几乎不变，表明注射Scop后的小鼠学习能力显著下降。WGOS40+Scop组和WGOS80+Scop组小鼠平均逃逸潜伏期接近盐水对照组（P﹥0.05）但明显低于Scop组（P﹤0.01），表明WGOS能显著改善由Scop诱导的学习获得障碍。2﹑在Morris水迷宫的定位航行测试中，单独注射盐水与WGOS40、WGOS80的小鼠，其平均逃逸潜伏期﹑总路程无显著性差异（P﹥0.05），表明单独注射WGOS不改变正常小鼠的记忆情况。Scop组小鼠的平均逃逸潜伏期﹑总路程明显高于盐水对照组（P＜0.01），表明注射Scop后的小鼠记忆能力显著下降。WGOS40+Scop组和WGOS80+Scop小鼠平均逃逸潜伏期﹑总路程接近盐水对照组（P﹥0.05）但明显低于Scop组（P﹤0.01），表明WGOS能显著改善由Scop诱导的记忆障碍。3﹑在Morris水迷宫的空间搜索测试中，我们测定了穿过平台次数、经过有效区次数、总路程等指标。结果发现，单独注射盐水与WGOS40、WGOS80的小鼠间无显著性差异（P﹥0.05），表明单独注射WGOS不改变正常小鼠的空间记忆情况。Scop组小鼠上述指标均明显高于盐水对照组（P＜0.01），表明注射Scop后的小鼠空间记忆能力显著下降。WGOS40+Scop组和WGOS80+Scop组小鼠上述指标接近盐水对照组（P﹥0.05）但明显低于Scop组（P﹤0.01），表明WGOS能显著改善由Scop诱导的空间记忆障碍。4﹑行为测试后，我们测定了小鼠海马组织的ACh含量。结果表明:单独注射盐水与WGOS40、WGOS80的小鼠海马中ACh含量无显著性差异（P﹥0.05），表明单独注射WGOS不改变正常小鼠海马中ACh含量。Scop组小鼠海马中ACh含量低于盐水对照组（P﹤0.05），而WGOS40+Scop组和WGOS80+Scop组小鼠海马中ACh含量接近盐水对照组（P﹥0.05）但明显高于Scop组（P﹤0.05），表明WGOS能显著改善由Scop诱导的海马ACh含量降低的状况。5﹑我们也测定了小鼠海马中AChE活性。结果表明：单独注射Saline与WGOS40、WGOS80的小鼠海马中AChE活性无显著性差异（P﹥0.05），表明单独注射WGOS不改变正常小鼠海马中AChE活性。Scop组小鼠海马中AChE活性明显高于盐水对照组（P﹤0.05）。WGOS40+Scop组和WGOS80+Scop组小鼠海马中AChE活性接近盐水对照组（P﹥0.05）但明显低于Scop组（P﹤0.05），表明WGOS能显著改善由Scop诱导的AChE活性升高的状况。6﹑我们还测定了小鼠海马中CAT活性。结果表明：单独注射Saline与WGOS40、WGOS80的小鼠海马中CAT活性无显著性差异（P﹥0.05），表明单独注射WGOS不改变正常小鼠海马中CAT活性。Scop组小鼠海马中CAT活性明显低于盐水对照组（P﹤0.01），WGOS40+Scop组和WGOS80+Scop组小鼠海马中CAT活性接近盐水对照组（P﹥0.05）但明显高于Scop组（P﹤0.05），表明WGOS能显著改善由Scop诱导的CAT活性降低的状况。由此，我们得出如下结论：WGOS能明显改善Scop诱导的空间学习记忆缺陷，但不影响正常小鼠的学习记忆过程。其机制涉及：①WGOS能够改善由Scop诱导的小鼠海马组织中AChE活性升高，进而恢复ACh含量，改善空间学习记忆能力。②W GOS能显著改善由Scop诱导的小鼠海马组织中CAT活性降低，减少自由基产生，减轻脑内氧化应激，防止神经细胞功能损伤，改善学习记忆能力。

    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.

水溶性人参寡糖改善东莨菪碱诱导的学习障碍及相关机制研究

前言4-6中文摘要6-13Abstract13-23第1章 文献综述29-45    1.1 学习记忆的生理机制及认知障碍29-39        1.1.1 胆碱能系统参与学习记忆过程30-33        1.1.2 氧化性应激也被涉及老年痴呆症的发病过程33-35        1.1.3 海马是学习记忆的重要核团35-38        1.1.4 评价实验动物学习记忆功能的常用方法38-39    1.2 人参及其生物活性成分的药理性作用39-45第2章 实验材料与方法45-55    2.1 实验材料45-46        2.1.1 主要试剂45        2.1.2 主要仪器设备45-46        2.1.3 实验动物46    2.2 实验方法46-51        2.2.1 实验动物药物处理46-47        2.2.2 Morris 水迷宫测试47-50        2.2.3 组织样品的收集50-51    2.3 生化检测51-54        2.3.1 乙酰胆碱 ACh 含量测定51-52        2.3.2 乙酰胆碱酯酶 AChE 活性测定52-53        2.3.3 过氧化氢酶 CAT 活性测定53        2.3.4 总蛋白浓度的测定53-54    2.4 数据处理54-55第3章 实验结果55-64    3.1 连续应用 WGOS 的小鼠体重明显增加55-56    3.2 Scop 诱导的空间学习记忆障碍小鼠模型的制备56    3.3 WGOS 改善 Scop 诱导的空间学习记忆障碍的行为学分析56-61        3.3.1 WGOS 对 Scop 诱导的学习障碍小鼠水迷宫训练的影响56-57        3.3.2 WGOS 对 Scop 诱导的学习障碍小鼠水迷宫定位航行测试的影响57-59        3.3.3 WGOS 对 Scop 诱导的学习障碍小鼠水迷宫空间搜索测试的影响59-61    3.4 WGOS 对 Scop 诱导的学习障碍小鼠海马中胆碱能系统的影响61-63        3.4.1 WGOS 改善 Scop 诱导的学习障碍小鼠海马中 ACh 含量61-62        3.4.2 WGOS 改善 Scop 诱导的学习障碍小鼠海马中 AChE 活性62-63    3.5 WGOS 改善 Scop 诱导的学习障碍小鼠海马中 CAT 活性63-64第4章 讨论64-73第5章 结论73-74参考文献74-89作者简介及在学期间所取得的科研成果89-90致谢90-91

  本文关键词：水溶性人参寡糖改善东莨菪碱诱导的学习障碍及相关机制研究，由笔耕文化传播整理发布。