基于突触可塑性的加味四逆散对睡眠剥夺大鼠学习记忆障碍的调控机制研究

【作者】 范新六；

【导师】 李峰；

【作者基本信息】 北京中医药大学 ， 中医诊断学， 2011， 博士

【摘要】 学习和记忆是脑的最高机能——智能的重要组成部分。学习指个体对新知识的获得过程,而记忆是指对这种获得知识的编码、巩固、检索,或者说记忆是刺激消失后在心理上保持下来的代表原刺激的讯息。睡眠和学习记忆能力关系密切。而睡眠障碍已成为困扰当代人群日常工作、学习和生活的一种常见因素。因此,本课题以睡眠剥夺复制学习记忆障碍模型,观察加味四逆散调控模型动物学习记忆能力的神经生物学机制。上篇理论研究对中医经典文献、历代医家专著和期刊文献涉及学习记忆的资料,进行整理、分析、归纳和总结。从五藏神与学习记忆的关系入手研究,侧重论述了心、肝和肾在学习与记忆过程中的作用。心神、肝魂和肾志调控学习记忆过程,依赖于心主血脉、肝主疏泄和肾藏精的功能或物质基础。在心神“任物“的主宰下,肝魂和肾志神相互作用、相互影响调控学习的获得、记忆的储存和再现过程。睡眠一觉醒周期受五藏神的调控,而睡眠剥夺主要影响了心主神明、肝藏魂和肾藏志的功能,导致学习记忆能力障碍。下篇实验研究实验研究一SD大鼠一般状况及学习记忆成绩的评价方法：应用Y迷宫评价各组大鼠学习记忆能力的状况,并对各组一般状况进行观察和记录。结果：①体重变化：在SD72h和168h时点,与模型组比较,四逆散组和加味四逆散组大鼠体重减轻量有统计学差异(p<0.05)；与其余四组体重量呈现减少的表现相反,对照组大鼠体重量呈现增加的表现。②血清酶学变化：模型组大鼠血清LDH较对照组明显增多(p<0.05)。加味四逆散组血清LDH较模型组明显降低(p<0.01)。③SD168h后,大鼠Y迷宫正确百分率较造模前明显下降(p<0.05),与对照组比较也明显下降(p<0.05)。在三个中药复方中,加味四逆散能明显增加模型大鼠迷宫成绩(p<0.05),而四逆散、生慧汤对模型大鼠迷宫成绩无明显变化。结论：①SD导致大鼠学习记忆能力下降,并出现躯体疲劳；②加味四逆能明显改善SD致学习记忆能力障碍,而四逆散和生慧汤对SD致学习记忆能力障碍改变不明显实验研究二SD大鼠行为学变化及加味四逆散对其调控作用方法：应用旷场和高架十字迷宫评价各组大鼠精神行为的变化。结果：①OFT变化：在水平活动上,各组大鼠SD168h后,模型组在总穿格次数上明显高于对照组(p<0.01)；模型组在中央格停留时间明显高于对照组(p<0.01)；模型组在中央格停留次数明显高于对照组(p<0.05),而生慧汤组和加味四逆散组明显低于模型组(p<0.05)。在垂直活动上,模型组站立次数明显高于对照组(p<0.01),加味四逆散组明显低于模型组(p<0.01)；生慧汤组也低于模型组(p<0.05)。②EPM变化：在水平活动上,模型组在进入开放臂时间百分比上明显高于对照组(p<0.05),而加味四逆散组明显低于模型组(p<0.05)。在垂直活动上,模型组向下探究时间明显高于对照组(p<0.05)。结论：①SD大鼠减少了焦虑样行为和增强了探索行为。②加味四逆散能显著改善SD所致大鼠焦虑样行为减少和探索行为增强的异常变化。生慧汤和四逆散仅能部分改善SD所致大鼠焦虑样行为减少和探索行为增强的异常变化。实验研究三SD大鼠PFC和海马CA3区突触界面结构的变化及加味四逆散对其调节作用方法：采用透射电镜技术,观察各组大鼠PFC和海马CA3区神经元突触界面超微结构的变化。结果：①PFC神经元突触界面超微结构的变化：与对照组比较,模型组大鼠PFC神经元突触间隙明显变宽(P<0.05),PSD厚度明显变薄(P<0.05)。与模型组比较,加味四逆散组大鼠PFC神经元、PSD厚度明显变厚(P<0.05)。②海马CA3区神经元突触界面超微结构的变化：与对照组比较,模型组大鼠海马CA3区神经元突触PSD厚度明显变薄(P<0.05),突触间隙厚度变宽(P<0.05)。与模型组比较,四逆散组和生慧汤组大鼠海马CA3区神经元突触间隙厚度明显变窄(P<0.05)。与模型组比较,加味四逆散组大鼠海马CA3区神经元PSD厚度明显增厚(P<0.05),突触间隙厚度明显变窄(P<0.05)。结论：①加味四逆散能增加SD大鼠PFC和海马CA3区神经元突触PSD厚度、调整海马CA3区突触间隙宽度,影响突触结构可塑性,增加突触传递效能。②四逆散和生慧汤仅能调整海马CA3区神经元突触间隙宽度,对突触传递效能影响低于加味四逆散组。实验研究四SD大鼠PFC和海马CA3区NMDA和EphB2受体蛋白质表达的变化及加味四逆散对其调节作用方法：采用Western blot技术,观察各组大鼠PFC和海马CA3区NMDA和EphB2受体蛋白质表达的变化。结果：①NMDA受体NR2A亚基蛋白质表达变化：与对照组比较,SD168h后大鼠PFC脑区NMDA受体MNR2A亚基蛋白质的表达明显减少(p<0.05)。与模型组比较,加味四逆散组大鼠PFC脑区NMDA受体MNR2A亚基蛋白质的表达明显增多(p<0.05)。与四逆散组比较,加味四逆散组大鼠PFC脑区NMDA受体MNR2A亚基蛋白质的表达明显增多(p<0.05)。②NMDA受体NR2B亚基蛋白质表达变化：与对照组比较,SD168h后,大鼠PFC和海马CA3区NMDA受体NR2B蛋白质的表达明显减少(p<0.05)。与模型组比较,加味四逆散组大鼠PFC和海马CA3区NMDA受体NR2B蛋白质的表达均明显增多(p<0.05)。与四逆散组比较,加味四逆散组大鼠PFC脑区NMDA受体NR2B蛋白质的表达均明显增多(p<0.05)。③NMDA受体NR2B (Tyr1472)蛋白质表达变化：与对照组比较,SD168h后大鼠PFC脑区NMDA受体NR2B (Tyr1472)蛋白质的表达明显减少(p<0.05)。与模型组比较,加味四逆散组大鼠PFC脑区NMDA受体NR2B (Tyr1472)蛋白质的表达均明显增多(p<0.05)。各组大鼠海马CA3区NMDA受体NR2B (Y1472)蛋白质的表达比较均无明显变化。④EphB2受体蛋白质表达变化：与对照组比较,SD168h后,大鼠PFC脑区EphB2受体蛋白质的表达明显减少(p<0.05)。与模型组比较,四逆散组、生慧汤组、加味四逆散组大鼠PFC脑区EphB2受体蛋白质的表达表达均明显增加(p<0.05)。各组大鼠海马CA3区EphB2受体蛋白质的表达比较均无明显变化。结论：①加味四逆散能上调SD大鼠PFC区的学习记忆相关受体NR2B、NR2B (Tyr1472)和EphB2蛋白质的表达；并上调海马CA3区NR2B受体蛋白质的表达。②四逆散和生慧汤仅能上调SD大鼠PFC区的EphB2蛋白质的表达。实验研究五SD大鼠海马CA1区NMDA和EphB2受体基因表达的变化及加味四逆散对其调节作用方法：采用实时荧光定量PCR技术,观察各组大鼠海马CA1区中枢神经受体基因表达的变化。结果：与对照组比较,模型组大鼠海马CA1区NMDA受体NR2B亚基和EphB2的mRNA表达明显减少(p<0.01), NMDA受体NR2A基因表达无明显变化。与模型组比较,加味四逆散组能明显增加大鼠海马CA1区NMDA受体NR2A, NR2B和EphB2的mRNA表达(p<0.01)。与模型组比较,生慧汤组能明显增加大鼠海马CA1区NMDA受体NR2A的mRNA表达(p<0.01)。结论：加味四逆散能明显上调SD大鼠海马CA1区学习记忆相关中枢神经受体基因表达的变化,而四逆散和生慧汤对其影响不明显。小结对研究数据进行分析,可以总结为四点：一是加味四逆散(疏肝补肾养心法)对SD大鼠从行为学、形态学、基因表达、蛋白质表达等多个层面进行调控,能明显改善SD大鼠学习记忆成绩。四逆散(疏肝法)和生慧汤(补肾养心法)仅影响SD大鼠在形态学、蛋白质表达等部分层面的部分靶点,对SD大鼠学习记忆成绩改变无统计学上的影响。二是PFC、海马CA1和CA3亚区相比较来说,加味四逆散对SD大鼠PFC区影响最为明显,特别是对PFC区学习记忆相关受体蛋白质表达的变化。三是加味四逆散(疏肝补肾养心法)改善SD大鼠学习记忆能力障碍,与增加SD大鼠PFC和海马CA3神经元PSD厚度,增加PFC区NMDA受体NR2B亚基和NR2B (Y1472)蛋白质的表达,上调海马CA3区NMDA受体NR2B亚基和EphB2受体蛋白质的表达,上调海马CA1区NR2B和EphB2的mRNA的表达有关。四是疏肝法和补肾养心法都参与了学习记忆过程的调控,是调控学习记忆过程的重要参与者,但不能独立发挥作用。更多还原

【Abstract】 Learning and memory is the highest function of the brain——an important component of intelligence. To Individual, learning refers to the process of acquisition of new knowledge. Memory is on access to knowledge encoding, consolidation, retrieval. In advanced animals and humans, learning and memory are the most important kind of method in order to adapt to the environment. Sleep is closely related to learning and memory.ln this subject, the researcher would observe the changes of the ability of learning and memory of sleep deprived rats and the effect of three Chinese formulas.PartⅠthe Theoretical StudyBy literature study, the relation of Chinese Five Viscera containing God.and learning and memory was discussed.The narrow god of Chinese medicine can be divided into five directional gods and Emotions. The God of Chinese Five Viscera influence each other, and regulate the process of learning and memory. Sleep-wake cycle is controlled by the God of Chinese Five Viscera.Sleep deprivation can damage the function of the God of Chinese Five Viscera.PartⅡExperimental StudiesIn this part, we focus on the researches of mechanism of molecular biology of learning and memory capacity changes caused by Sleep deprivation. The learning and memory capacity through establishing animal models of Sleep deprivation was evaluated. Using western blot and fluorescence quantitative PCR methods, the changes of protein and gene xpression of NMDA and EphB2 receptors in different brain regions of model rats were observed. The changes of the ultrastructure of rat prefrontal cortex and hippocampus were observed using transmission electron microscopy. Different Chinese herbs were used to intervene for animal models, and their roles in medicine mechanisms were investigated. Main content and the results are as follows:Experiment One:Evaluation of the Abilities of Learning and Memory of Sleep Deprived Rats.Objective:To observe the changes of the ability of learning and memory of sleep deprived rats and the effect of three Chinese formulas.Methods:Changes of learning and memory capacity were observed using Y maze.Results:Compared with the control group, the Y maze correct percentage in the model group obviously decreased (P<0.05). Compared with the model group, the Jiawei Sini Powder group can obviously increase the Y maze correct response rate(P<0.05).Conclusion:Jiawei Sini Powder could improve sleep deprived rats’ability of learning and memory.Experiment Two:Study of Effect of Jiawei Sini Powder on the Changes of behaviour of Sleep Deprived Rats.Objective:To investigate the behavioral changes of 168h of sleep deprived rats and the effect of three Chinese formulas(Sini Powder, Shenghui Decoction and Jiawei Sini Powder).Methods:The rats were deprived sleep for 168 hours using multi-platform method. Experimental rats were randomly divided into the control group, the model group, the Sini Powder group, the Shenghui Decoction group and the Jiawei Sini Powder group, fifteen in each.Corresponding medicines and distilled water were given to them every day.The intervention groups were fed with Sini Powder,Shenghui Decoction and jiawei Sini Powder, and the dosages are 2.5g/kg-1,13.75g/kg-1,16.25g·kg-1.To observe the behavioral changes of rats using open field and elevated plus-maze test.Results:After 168h of sleep deprivation, rats made significantly more entries into the open arms and inner zone than control group in the elevated plus maze and standard open field tests. Rats of Jiawei Sini Powder group spent significantly less time in the open arms and inner zone than sleep deprivation group.Conclusion:The study indicates Jiawei Sini Powder can significantly influence abnormal changes of anxiety-like behavior and explore behavior of sleep deprived rats.Experiment Three:Experimental Study of Effect of Jiawei Sini Powder on the Changes of Synaptic Plasticity in PFC and hippocampus in Sleep Deprived Rats.Objective:To observe the effect of jiawei Sini Powder on learning and memory and the changes of synaptic plasticity in PFC and hippocampal CA3 subfield in sleep deprived rats. Methods:The changes of the ultrastructure of rat prefrontal cortex was observed using transmission electron microscopy.Results:Compared with control group, the percentage of correct responses of model group was significantly decreased (P<0.05).The percentage of correct responses of Jiawei Sini Powder group was significantly higher than model group (P<0.05).Compared with control group, in PFC and hippocampal CA3 area of the model group,the thickness of PSD was significantly declined (P<0.05) and Synaptic cleft was significantly widened (P<0.05).Compared with model group, in hippocampal CA3 area of the Jiawei Sini Powder group,the thickness of PSD was significantly added (P<0.05) and Synaptic cleft was narrowed (P<0.05).Compared with model group, in PFC area of the Jiawei Sini Powder group,the thickness of PSD was significantly added (P<0.05)). Compared with model group, in hippocampal CA3 area of the Sini Powder group and Shenghui Decoction group,Synaptic cleft was significantly narrowed (P<0.05).Conclusion:By influencing synaptic Structural plasticity in PFC and hippocampal CA3 area, Jiawei Sini Powder can improve the ability of learning and memory in sleep deprived rats.Experiment Four:Effect of Jiawei Sini Powder on ProteinExpressions of PFC and Hippocampal CA3 area NMDA and EphB2 Receptor in Sleep Deprived RatsObjective:To study protein expressions of PFC and hippocampal CA3 area NMDA and EphB2 receptor in sleep deprived rats, and observe effects of Sini Powder, Shenghui Decoction, and Jiawei Sini Powder on them. Methods:Protein expressions of NR2A, NR2B, NR2B(Tyr1472) and EphB2 receptor in sleep deprived rats were quantitatively analyzed using Western blot technology.Results: Compared with the control group, protein expressions of NR2A, NR2B, NR2B(Tyr1472) and EphB2 receptor in PFC area obviously decreased (P< 0.05),and protein expressions of NR2B in hippocampal CA3 area aslo decreased(P <0.05). Compared with the model group, Jiawei Sini Powder group could obviously improve protein expressions of NR2A, NR2B, NR2B(Tyr1472) and EphB2 receptor in PFC area (P<0.05), and increase protein expressions of NR2B, in hippocampal CA3 area (P<0.05). Compared with the model group, both Sini Powder group and Shenghui Decoction group could obviously improve protein expressions of EphB2 receptor in PFC area (P<0.05).Conclusion:Sleep deprivation could reduce protein expressions of NR2B, NR2B(Tyr1472)and EphB2 receptor in PFC area, and decrease protein expressions of NR2B in hippocampal CA1 area. Jiawei Sini Powder could improve rats’ability of learning and memory, which might be possibly achieved through up-regulating protein expression of EphB2 and NR2B receptor, and changing the synaptic plasticity of hippocampus and PFC.Experiment Five:Effect of Jiawei Sini Powder on mRNAExpressions of Hippocampal CA1 area NMDA Subunit NR2A and NR2B, and EphB2 Receptor in Sleep Deprived RatsObjective:To study mRNA expressions of hippocampal N-methyl D-aspartate (NMDA)receptor subunit NR2A and NR2B, and EphB2 receptor in sleep deprived rats, and observe effects of Sini Powder, Shenghui Decoction, and Jiawei Sini Powder on them. Methods:Gene expressions of NMDA subunit NR2A and NR2B, and EphB2 receptor in sleep deprived rats were quantitatively analyzed using fluorescence quantitative PCR.Results:Compared with the control group,mRNA expressions of NR2B and EphB2 receptor in hippocampal CA1 area obviously decreased (P<0.01), with no obvious change of NR2A Compared with the model group, Jiawei Sini Powder group could obviously improve mRNA expressions of NR2A, NR2B and EphB2 receptor(P<0.01).No statistical difference was found between the Sini Powder group and the Shenghui Decoction group.In hippocampal CA1 area,mRNA expression of NR2A could be more obviously increased in the Shenghui Decoction group than in the model group (P<0.01).Conclusion:Sleep deprivation could reduce gene expressions of NR2B and EphB2 receptor in hippocampal CA1 area. Jiawei Sini Powder could improve rats’ ability of learning and memory, which might be possibly achieved through up-regulating mRNA expression of EphB2 and NR2B receptor in hippocampal CA1 area, and changing the synaptic plasticity of hippocampus.SummaryJiawei Sini Powder could improve rats’ ability of learning and memory, which might be possibly achieved through changing the synaptic plasticity of PFC and hippocampus. Smoothing Liver Qi and replenishing Kidney play an important role in the process of regulating learning and memory, but can’t independently operate this process. When nourishing Heart, smoothing Liver Qi and replenishing Kidney were taken together, the ability of learning and memory would be improved.更多还原