远志总皂苷对AD模型大鼠学习记忆及海马nAchRα7亚基的影响
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摘要
阿尔茨海默病(Alzheimer's disease,AD)已成为现代世界关注的热点问题之一。AD是老年人最常见的痴呆的原因，患病的风险随着年龄的增大而增大。“痴呆”包括认知、思维、推理和记忆能力的下降。目前还没有任何方法和药物可完全有效治愈AD，仅有一些药物可以暂时缓解症状或延缓疾病的进程。目前公认的胆碱能系统活性下降是引起AD的重要原因。很多研究明确表明AD患者脑中乙酰胆碱缺乏、烟碱型乙酰胆碱受体( nicotinicacetylcholine receptors, nAChRs)表达减少是常见的现象。实验表明缺乏nAChRα7亚基的小鼠与含有nAChRα7亚基小鼠的对照组相比，在水迷宫实验中学习记忆能力降低。
远志是一味益智强记的中药，远志总皂苷(Tenuigenin ,TEN)为其粗提取物。有实验表明TEN可以改善学习和记忆，但药理作用不是很清楚。目前有关TEN对海马区的nAChRα7的影响未见报道。
本实验拟采用D-半乳糖(D-galactose, D-gal)致衰联合Meynert基底核损毁建立AD大鼠模型，在此基础上用Morris水迷宫实验观察不同剂量的TEN对AD模型动物的学习记忆能力的影响并用免疫组化法不同剂量的TEN对AD模型动物海马CA1区nAChRα7的表达水平。探讨TEN改善学习记忆的可能的机制。
第一部分远志总皂苷对AD模型大鼠学习记忆的影响
目的：①观察TEN对AD模型大鼠学习记忆的影响；②该影响是否具有剂量依赖性。
方法：32只健康雄性Wistar大鼠随机分为对照组、AD模型组、TEN低剂量组和TEN高剂量组，每组8只，采用Morris水迷宫实验来检测各组大鼠的学习记忆能力。
结果：①在5天的定位航行试验中，各组大鼠的逃避潜伏期（EL）均呈逐渐缩短并下降趋势；与对照组相比，TEN高剂量组平均逃避潜伏期（EL）显著延长(P<0.05)；与模型组相比，TEN高、低剂量组平均逃避潜伏期（EL）明显缩短(P<0.05)；与对照组相比，模型组大鼠平均逃避潜伏期（EL）明显延长(P<0.05)。②通过对第6天空间探索实验各组大鼠的跨越原平台的次数和平台象限停留的时间的比较发现：与对照组比较，模型组大鼠的跨越原平台的次数明显减少（P<0.05），平台象限停留时间明显缩短（P<0.05）；与模型组比较，TEN低、高剂量组大鼠跨越原平台的次数明显增加（P<0.05），在平台象限停留时间明显增加（P<0.05）；与TEN低剂量组比较，TEN高剂量组大鼠跨越原平台的次数显著延长（P<0.05），在平台象限停留时间明显延长（P<0.05）。
结论：TEN能改善AD模型大鼠的学习记忆能力，且该作用可能具有剂量依赖性。
第二部分远志总皂苷对AD模型大鼠海马nAChRα7亚基的影响
目的：①观察TEN对AD模型大鼠海马CA1区烟碱型乙酰胆碱受体α7亚基的影响，探索其抗AD的作用机制；②该影响是否具有剂量依赖性。
方法：分组同第一部分，水迷宫实验结束后，动物心脏灌注、固定、取脑、切片，用SABC染色法，进行免疫组化实验。观察海马CA1区烟碱型乙酰胆碱受体α7亚基的表达水平的改变。
结果：与对照组相比，模型组海马CA1区nAChRα7的表达水平明显下降(P<0.05)；与模型组相比，TEN高、低剂量组大鼠海马CA1区nAChRα7的表达水平较明显增加(P<0.05)；与TEN低剂量组相比，TEN高剂量组海马CA1区nAChRα7的表达水平较明显增加(P<0.05)。
结论：TEN可显著提高AD模型大鼠海马CA1区nAChRα7的表达且具有剂量依赖性，这可能是TEN改善学习记忆和认知功能的部分机制。
Alzheimer's disease (AD) has become a more and more hot issue in modern society with theacceleration of population senescence in the world.
Alzheimer's disease (AD) is the most common cause of dementia among older people. Therisk goes up as you get older,The term 'dementia' describes a set of symptoms which can includea decline in cognitive function or mental ability,thinking,reasoning and remembering.Alzheimer's is a progressive disease, Currently there is no cure for Alzheimer's disease. However,drug treatments can temporarily alleviate some symptoms or slow down their progression insome people. Many individuals with Alzheimer's have been shown to have a shortage of thechemical acetylcholine in their brains.The decreased activity of cholinergic system is Currently recognized,which is one of important reasons for the cause of AD. The expression of nicotinicacetylcholine receptor (nicotinic acetylcholine receptors, nAChRs) in the brain of AD isdecreased,which is a common phenomenon.Experiments show that compared with the controlgroup,the memory of the rats which were the lack of nAChRα7 subunits was decreased in theMorris water maze test.
Tenuifolin(TEN) is a crude extract of Polygala tenuifolia Willd.which is commonly used intraditional Chinese herbal medicine for nootropic.Previous studies have shown that tenuigenin,can improve learning and memory ability. However, the mechanism underlying this effect isunknown.
The impact of TEN on the expression of Nicotinic Acetylcholine Receptor subunit alpha-7in hippocampus region of Alzheimer’s disease model has not been reported.
In this study，the model of Alzheimer’s disease was made by orientally injecting ibotenicacid into Meynert basal nuclei of aging rat induced by D-gal. Morris maze test was used toevaluate the learning and memory of each rat, Immunohistochemistry was performed to estimatethe changes of the expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus region of rats.
PartⅠEffects of Tenuigenin on Learning and Memory Of AD Rats
Objective: To observe the effects of TEN on learning and memory of AD rats caused byD-gal and IBO, and to prove whether the effects are dose-dependent.
Methods: 32 male Wistar rats were randomized into control group, model group, high-dosegroup and low-dose group. The model of Alzheimer’s disease was made by orientally injectingibotenic acid into Meynert basal nuclei of aging rat induced by D-gal Morris maze test was usedto evaluate the learning and memory of each rat.
Results:①In the five days of the place navigation test, the escape latency of each rats weregradually decreased;Compared with control group,the average escape latency of the high doseTEN group were significantly prolonged(P<0.05).Compared with model group,the averageescape latency of the high and low dose TEN group were significantlyshortened(P<0.05);Compared with control group,the average escape latency of the model groupwere significantly prolonged(P<0.05).②Compared with control group, the times of passingplatform was significantly decreased, (P<0.05）and the time of staying at platform quad wassignificantly shortened（P<0.05）in model group; Compared with model group, the times ofpassing platform were significantly increased（P<0.05）and the time of staying at platform quadwere significantly prolonged（P<0.05）, in both high dose group and low dose group; Furthermore, Compared with low dose group,the time of staying at platform quad and the timesof passing platform was significantly increased（P<0.05）in high dose group.
Conclusion: This data suggests that TEN can improve learning and memory ability of ADrats potentially dose-dependently.
PartⅡEffects of Tenuigenin on the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus Of AD RatsObjective: To observe the effects of TEN on the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus CA1 region Of AD Rats caused by D-gal and IBO.Toinvestigate the mechanism underlying the effects of TEN on learning and memory; and to provewhether the effects are dose-dependent.
Methods: The group assigned as PartⅠ. Perfusion,solidification,takeing the brain,slicing ofspecimen and immunohistochemistry（SABC）were performed to estimate the changes of theexpression of Nicotinic Acetylcholine Receptor subunit alpha-7 in hippocampus CA1 region ofratsAfter Morris maze test.
Results: Compared with control group, the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus CA1 region of model group obviouslydeclined(P<0.05). The expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus CA1 region in high and low dose Tenuigenin treatment group decreasedsignificantly(P<0.05)compared with the model group. Compared with the low dose Tenuigenintreatment group, the expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus CA1 region in high dose Tenuigenin treatment group increasedsignificantly(P<0.05).
Conclusion: This data suggests that TEN can potentially dose-dependently increase theexpression of Nicotinic Acetylcholine Receptor subunit alpha-7 in hippocampus CA1 region ofAlzheimer’s disease model rats, which may partly explain the beneficial effects of TENon learning and memory and cognitive function.
远志是一味益智强记的中药，远志总皂苷(Tenuigenin ,TEN)为其粗提取物。有实验表明TEN可以改善学习和记忆，但药理作用不是很清楚。目前有关TEN对海马区的nAChRα7的影响未见报道。
本实验拟采用D-半乳糖(D-galactose, D-gal)致衰联合Meynert基底核损毁建立AD大鼠模型，在此基础上用Morris水迷宫实验观察不同剂量的TEN对AD模型动物的学习记忆能力的影响并用免疫组化法不同剂量的TEN对AD模型动物海马CA1区nAChRα7的表达水平。探讨TEN改善学习记忆的可能的机制。
第一部分远志总皂苷对AD模型大鼠学习记忆的影响
目的：①观察TEN对AD模型大鼠学习记忆的影响；②该影响是否具有剂量依赖性。
方法：32只健康雄性Wistar大鼠随机分为对照组、AD模型组、TEN低剂量组和TEN高剂量组，每组8只，采用Morris水迷宫实验来检测各组大鼠的学习记忆能力。
结果：①在5天的定位航行试验中，各组大鼠的逃避潜伏期（EL）均呈逐渐缩短并下降趋势；与对照组相比，TEN高剂量组平均逃避潜伏期（EL）显著延长(P<0.05)；与模型组相比，TEN高、低剂量组平均逃避潜伏期（EL）明显缩短(P<0.05)；与对照组相比，模型组大鼠平均逃避潜伏期（EL）明显延长(P<0.05)。②通过对第6天空间探索实验各组大鼠的跨越原平台的次数和平台象限停留的时间的比较发现：与对照组比较，模型组大鼠的跨越原平台的次数明显减少（P<0.05），平台象限停留时间明显缩短（P<0.05）；与模型组比较，TEN低、高剂量组大鼠跨越原平台的次数明显增加（P<0.05），在平台象限停留时间明显增加（P<0.05）；与TEN低剂量组比较，TEN高剂量组大鼠跨越原平台的次数显著延长（P<0.05），在平台象限停留时间明显延长（P<0.05）。
结论：TEN能改善AD模型大鼠的学习记忆能力，且该作用可能具有剂量依赖性。
第二部分远志总皂苷对AD模型大鼠海马nAChRα7亚基的影响
目的：①观察TEN对AD模型大鼠海马CA1区烟碱型乙酰胆碱受体α7亚基的影响，探索其抗AD的作用机制；②该影响是否具有剂量依赖性。
方法：分组同第一部分，水迷宫实验结束后，动物心脏灌注、固定、取脑、切片，用SABC染色法，进行免疫组化实验。观察海马CA1区烟碱型乙酰胆碱受体α7亚基的表达水平的改变。
结果：与对照组相比，模型组海马CA1区nAChRα7的表达水平明显下降(P<0.05)；与模型组相比，TEN高、低剂量组大鼠海马CA1区nAChRα7的表达水平较明显增加(P<0.05)；与TEN低剂量组相比，TEN高剂量组海马CA1区nAChRα7的表达水平较明显增加(P<0.05)。
结论：TEN可显著提高AD模型大鼠海马CA1区nAChRα7的表达且具有剂量依赖性，这可能是TEN改善学习记忆和认知功能的部分机制。
Alzheimer's disease (AD) has become a more and more hot issue in modern society with theacceleration of population senescence in the world.
Alzheimer's disease (AD) is the most common cause of dementia among older people. Therisk goes up as you get older,The term 'dementia' describes a set of symptoms which can includea decline in cognitive function or mental ability,thinking,reasoning and remembering.Alzheimer's is a progressive disease, Currently there is no cure for Alzheimer's disease. However,drug treatments can temporarily alleviate some symptoms or slow down their progression insome people. Many individuals with Alzheimer's have been shown to have a shortage of thechemical acetylcholine in their brains.The decreased activity of cholinergic system is Currently recognized,which is one of important reasons for the cause of AD. The expression of nicotinicacetylcholine receptor (nicotinic acetylcholine receptors, nAChRs) in the brain of AD isdecreased,which is a common phenomenon.Experiments show that compared with the controlgroup,the memory of the rats which were the lack of nAChRα7 subunits was decreased in theMorris water maze test.
Tenuifolin(TEN) is a crude extract of Polygala tenuifolia Willd.which is commonly used intraditional Chinese herbal medicine for nootropic.Previous studies have shown that tenuigenin,can improve learning and memory ability. However, the mechanism underlying this effect isunknown.
The impact of TEN on the expression of Nicotinic Acetylcholine Receptor subunit alpha-7in hippocampus region of Alzheimer’s disease model has not been reported.
In this study，the model of Alzheimer’s disease was made by orientally injecting ibotenicacid into Meynert basal nuclei of aging rat induced by D-gal. Morris maze test was used toevaluate the learning and memory of each rat, Immunohistochemistry was performed to estimatethe changes of the expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus region of rats.
PartⅠEffects of Tenuigenin on Learning and Memory Of AD Rats
Objective: To observe the effects of TEN on learning and memory of AD rats caused byD-gal and IBO, and to prove whether the effects are dose-dependent.
Methods: 32 male Wistar rats were randomized into control group, model group, high-dosegroup and low-dose group. The model of Alzheimer’s disease was made by orientally injectingibotenic acid into Meynert basal nuclei of aging rat induced by D-gal Morris maze test was usedto evaluate the learning and memory of each rat.
Results:①In the five days of the place navigation test, the escape latency of each rats weregradually decreased;Compared with control group,the average escape latency of the high doseTEN group were significantly prolonged(P<0.05).Compared with model group,the averageescape latency of the high and low dose TEN group were significantlyshortened(P<0.05);Compared with control group,the average escape latency of the model groupwere significantly prolonged(P<0.05).②Compared with control group, the times of passingplatform was significantly decreased, (P<0.05）and the time of staying at platform quad wassignificantly shortened（P<0.05）in model group; Compared with model group, the times ofpassing platform were significantly increased（P<0.05）and the time of staying at platform quadwere significantly prolonged（P<0.05）, in both high dose group and low dose group; Furthermore, Compared with low dose group,the time of staying at platform quad and the timesof passing platform was significantly increased（P<0.05）in high dose group.
Conclusion: This data suggests that TEN can improve learning and memory ability of ADrats potentially dose-dependently.
PartⅡEffects of Tenuigenin on the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus Of AD RatsObjective: To observe the effects of TEN on the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus CA1 region Of AD Rats caused by D-gal and IBO.Toinvestigate the mechanism underlying the effects of TEN on learning and memory; and to provewhether the effects are dose-dependent.
Methods: The group assigned as PartⅠ. Perfusion,solidification,takeing the brain,slicing ofspecimen and immunohistochemistry（SABC）were performed to estimate the changes of theexpression of Nicotinic Acetylcholine Receptor subunit alpha-7 in hippocampus CA1 region ofratsAfter Morris maze test.
Results: Compared with control group, the expression of Nicotinic AcetylcholineReceptor subunit alpha-7 in hippocampus CA1 region of model group obviouslydeclined(P<0.05). The expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus CA1 region in high and low dose Tenuigenin treatment group decreasedsignificantly(P<0.05)compared with the model group. Compared with the low dose Tenuigenintreatment group, the expression of Nicotinic Acetylcholine Receptor subunit alpha-7 inhippocampus CA1 region in high dose Tenuigenin treatment group increasedsignificantly(P<0.05).
Conclusion: This data suggests that TEN can potentially dose-dependently increase theexpression of Nicotinic Acetylcholine Receptor subunit alpha-7 in hippocampus CA1 region ofAlzheimer’s disease model rats, which may partly explain the beneficial effects of TENon learning and memory and cognitive function.
引文
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[1]盛树力.中医药治疗老年性痴呆大有可为.亚太传统医药，2006，1l(1l):22．
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[9]王宇卉，邵福源，深化对认知功能障碍的认识，医药专论，2010,31（7）:385-389．
[10]王雪松，程兴旺．阿尔茨海默病的治疗进展（综述）．中国城乡企业卫生．2010，（3）：33-34．
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[13]任娟莉，袁若华，《千金要方》抗痴呆方药探析．现代中医药，2007，27（6）:52-53．
[14]邓家刚,郝二伟,郭宏伟等．老年性痴呆复方用药规律探讨．山东中医杂志2007，26（6）:363-365．
[15]王晓林，巴哈尔·哈德尔，明清时期治疗老年性痴呆的用药规律．光明中医，2010,25（5）:749-750．
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[22]Lopez OL，Hamilton RL, Becker JT, et al．Severity of cognitive impairment and the clinical diagnosis ofAD with Lewy Bodies．Neurology，2000,54(9)：1 780-l 787．
[23]Piccardi M, Congiu D, Squassina A, et a1．Alzheimer s disease：case—control association study ofpolymorphisms in ACHE，CHAT，and BCHE genes in a Sardinian sample．Am J Med Genet BNeuropsychiatr Genet，2007,144(7)：895—899．
[24]Michael E.Hasselmo．The Role of Acetyleholine in Learning and Memory. Curr OP Neurobiol，2006，16(6):710-715.
[25]穆俊霞,，李新毅.，聪脑汤对AD大鼠学习记忆和胆碱酯酶活性的影响.山西中医学院学报, 2006,7( 1 ): 21- 22.
[26]杨晓娟,刘田福,张生.健脑益智汤对类阿尔茨海默病大鼠血清胆碱酯酶和脑皮质细胞凋亡的影响[ J ].中国新药杂志, 2008, 17( 1): 32.
[27]穆俊霞，李新毅，中药远志对阿尔茨海默病大鼠模型学习记忆和胆碱酯酶活性的影响．世界中西医结合杂志，2007,2（1）:18-20．
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