四逆散加味对睡眠剥夺大鼠学习记忆影响的分子生物学机制研究

【作者】 冯婷；

【导师】 李峰；

【作者基本信息】 北京中医药大学 ， 中医诊断学， 2012， 博士

【摘要】 学习记忆力减退属中医“健忘”、“善忘”的范畴,是脑功能失常的重要体现,伴随生活压力的增大,学习记忆力下降也成为影响人们生活质量的重要因素之一。记忆下降由多种因素导致,在中青年人群中多因不良生活习惯引发,如缺乏运动、熬夜、嗜酒等,其中被广泛关注的是睡眠障碍引起的记忆力下降,这种睡眠障碍可通过剥夺睡眠模拟。现本课题以睡眠剥夺法复制学习记忆障碍模型,观察四逆散加味对模型动物学习记忆能力影响的神经生物学机制。第一部分理论研究对历代医家在睡眠与学习记忆方面的认识进行理论探讨,睡眠及学习记忆的形成和巩固与肝、心、肾的联系较大。在学习、记忆过程中,肝、心、肾、脑通过气血、经络、五藏神功能相互贯通,对信息处理各有分工。人对外界事物的接触首先从心的“任物”开始,对所感进行思考、分析,形成初步印象,经过回忆、存忆的过程进行再判断,并对信息进行整理,最后稳固记忆并存之于脑,整个过程贯穿肝的疏泄作用以及肝魂的调节。对于睡眠不足引起的学习障碍,理论上讲,肝失疏泄、心神失养和肾精不足可作为主要辨证类型,而不单单限于对心、肾的调节。第二部分实验研究研究一睡眠剥夺大鼠的一般情况及学习记忆能力评价目的：复制学习记忆障碍模型,通过行为学评价模型,并观察四逆散加味对学习记忆力的影响。方法：采用大鼠睡眠剥夺法复制学习记忆障碍模型,并观察各组大鼠的一般情况、体重、Y型迷宫成绩、血清SOD的变化。结果：①一般体征变化：从造模第3天开始到造模结束,对照组大鼠一般情况良好,模型组和用药组出现不同程度的躁狂、攻击性、警惕性高、撕咬、脱毛、便稀等体征,四逆散和复合方治疗组的体征较模型组和生慧汤治疗组减轻。②体重变化：造模开始到结束,观察第0、3、5、8天大鼠体重变化,对照组大鼠体重增加,模型组和三个用药组体重呈现不同程度减轻,与对照组比较,其余四组在4个时间点的体重都有非常显著差异,但四个组之间体重无差异。③Y型迷宫成绩：测试各组大鼠第0、7天的错误反应次数和主动回避率,第0天各组间无差异,造模第7天,错误反应次数结果显示,与对照组比较,模型组和三个用药组错误反应次数明显增多,模型组、四逆散治疗组、生慧汤治疗组有非常显著差异,复合方治疗组有显著差异；与模型组比较,复合方治疗组错误反应次数明显减少,有非常显著差异,而四逆散和生慧汤组无明显差异。主动回避率结果显示,与对照组比较,模型组、四逆散治疗组、生慧汤治疗组主动回避率降低,具有显著差异,而复合方治疗组无明显差异；与模型组比较,复合方治疗组主动回避率增加,有非常显著差异,四逆散治疗组增加,有显著差异。④血清SOD表达：与对照组比较,模型组和生慧汤治疗组SOD含量降低,有非常显著差异,四逆散和复合方治疗组SOD含量无显著差异；与模型组比较,四逆散和复合方治疗组SOD含量显著升高,两个治疗组之间比较,四逆散组显著低于复合方组。结论：①睡眠剥夺能导致大鼠记忆力下降,并伴随一般体征、体重的改变。②疏肝补肾养心方能改善睡眠剥夺所致的记忆障碍,疏肝方能部分改善记忆能力,单纯补肾养心无明显调节作用。研究二睡眠剥夺大鼠行为学的改变及四逆散加味的调控作用目的：观察四逆散加味对睡眠剥夺大鼠对新奇环境认知的影响。方法：应用自制旷场箱进行旷场实验。结果：造模第0天,各组旷场指标无显著差异。造模第7天,在直立次数(垂直得分)方面,与对照组比较,模型组明显减少,有非常显著差异；与模型组比较,三个治疗组次数增多,有显著差异。在穿格次数(水平得分)方面,与对照组比较,模型组明显减少；与模型组比较,四逆散治疗组增多,有显著差异,生慧汤治疗组明显增多,差异非常显著。造模第7天,中央区停留时间方面,与对照组比较,模型组时间增加,有非常显著差异；与模型组比较,生慧汤、复合方治疗组时间缩短,有显著差异,四逆散治疗组时间明显缩短,差异非常显著。在修饰次数上,与模型组相比,复合方治疗组明显增多,其余组间无显著差异。结论：①睡眠剥夺能降低大鼠的空间探索能力、兴奋性和认知能力,对适应能力无明显影响。②疏肝方、补肾养心方及复合方均可能改善睡眠剥夺所致的情绪、认知障碍,三个组方从不同角度进行调节。研究三睡眠剥夺对大鼠海马BDNF及高亲和力受体TrkB的基因和免疫阳性物表达的影响及四逆散加味的调节作用目的：观察四逆散加味对睡眠剥夺大鼠海马中学习记忆相关重要蛋白BDNF和TrkB表达的影响。方法：采用免疫组织化学和荧光定量PCR法,测定海马CA3区和DG区BDNF和TrkB蛋白免疫阳性物表达和海马中基因的表达。结果：①在海马CA3区和DG区均可见BDNF免疫阳性物的分布,其阳性产物为棕黄色颗粒状,呈椭圆形或锥形,主要分布在CA3区锥体细胞的胞浆、胞核(以核膜最密集)和DG区颗粒细胞的胞浆、胞核。TrkB免疫阳性物为细小的棕黄色颗粒,呈圆形或锥形,分布在CA3区锥体细胞的胞浆,胞核无分布。②BDNF蛋白表达：CA3区结果显示,与对照组比较,模型组和三个治疗组BDNF平均光密度值减小,差异非常显著；与模型组比较,复合方治疗组平均光密度值增大,差异非常显著,四逆散和生慧汤治疗组无显著差异。DG区结果显示,与对照组比较,模型组与三个治疗组的BDNF平均光密度值减小,差异非常显著；与模型组比较,四逆散、生慧汤和复合方治疗组的平均光密度值无显著差异。③BDNF基因表达：与对照组比较,模型组海马BDNF mRNA表达减少,差异非常显著；与模型组比较,四逆散和生慧汤治疗组BDNF mRNA表达无显著差异,复合方治疗组海马BDNF mRNA表达明显增多,差异非常显著。④TrkB蛋白表达：CA3区结果显示,与对照组比较,模型组和三个治疗组TrkB蛋白平均光密度值减小,差异非常显著；与模型组比较,四逆散和复合方治疗组的蛋白平均光密度值增加,差异非常显著,且四逆散组表达比复合方少。DG区结果显示,与对照组比较,模型组和三个用药组的TrkB阳性物平均光密度值减少,有非常显著差异；与模型组比较,三个用药组平均光密度值无显著差异。⑤TrkB基因表达：与对照组比较,模型组TrkB mRNA的表达减少,差异显著,四逆散、生慧汤、复合方治疗组无显著差异；与模型组比较,四逆散治疗组和复合方治疗组的TrkB mRNA表达量明显增多,差异显著,生慧汤治疗组的TrkB mRNA表达无明显增加。结论：①睡眠剥夺能降低海马BDNF和TrkB蛋白及基因的表达。②疏肝补肾养心方能上调海马BDNF和TrkB的基因表达和CA3区蛋白的表达。③疏肝方的作用点相对狭窄,对TrkB的表达有上调作用。研究四睡眠剥夺大鼠海马区非受体酪氨酸激酶Fyn基因表达及四逆散加味的干预作用目的：观察四逆散加味对睡眠剥夺大鼠海马Fyn基因表达的影响,总结Fyn与BDNF、TrkB在改善学习记忆方面的关联。方法：通过荧光定量PCR法,测定海马Fyn基因表达。结果：与对照组比较,模型组海马Fyn mRNA的表达量明显减少,具非常显著差异；与模型组比较,生慧汤、复合方治疗组大鼠海马Fyn mRNA的表达增多,以复合方治疗组增多明显,而四逆散治疗组无显著差异。结论：①睡眠剥夺能降低海马Fyn基因的表达。②补肾养心方和复合方能通过调节Fyn基因的表达改善睡眠剥夺形成的学习记忆力下降,复合方更明显。③睡眠剥夺大鼠海马Fyn与BDNF及其受体基因的表达具有一致性,三种蛋白对学习记忆的调节作用可能通过同一信号通路。研究五睡眠剥夺大鼠海马区ERK及pERK蛋白表达及四逆散加味的调节作用目的：观察四逆散加味对睡眠剥夺大鼠海马ERK及pERK蛋白表达的影响,并总结与非受体酪氨酸激酶及脑源性神经营养因子的联系。方法：通过免疫印迹法,观察ERK及其活化形式的蛋白表达。结果：①ERK2蛋白表达：与对照组比较,生慧汤与复合方治疗组表达增多,差异显著；模型组和四逆散治疗组未见明显变化。与模型组比较,生慧汤和复合方治疗组表达明显增多,差异显著。②pERK2蛋白表达：与对照组比较,模型组表达明显减少,复合方治疗组表达显著增多；与模型组比较,复合方和生慧汤治疗组都上调蛋白表达,以复合方的作用更明显。结论：①睡眠剥夺能明显减少记忆形成相关和pERK2的蛋白表达量,但对ERK2无显著作用。②复合方与补肾养心方能改善睡眠剥夺大鼠海马pERK的下降,上调ERK2及pERK2的表达,以pERK2的表达上调更明显。③ERK及其活化形式可能与BDNF、Fyn共同参与睡眠剥夺所致的学习记忆力下降。小结本研究首先据睡眠理论、五藏神理论,着重从肝、心、肾角度探讨睡眠的形成及学习记忆的发生机制,人对新事物的接触首先从心的“任物”开始(神),对所感进行思考、分析,形成初步印象(志),经过回忆、存忆的过程进行再判断,并对信息进行整理,稳固记忆并存之于脑。记忆的形成和巩固都与肝的疏泄以及肝魂调节作用有关,五藏神尤其是肝、心、肾对睡眠及引起的学习记忆障碍至关重要。分析实验研究结果总结为以下4点：①对大鼠进行连续7天睡眠剥夺可导致学习记忆障碍。②四逆散和生慧汤的复合方能通过调节海马内ERK-pERK/BDNF/TrkB/Fyn信号转导途径改善睡眠剥夺所致学习记忆障碍。③四逆散、生慧汤作用点较狭窄,从行为学、部分蛋白、基因几方面来调节记忆力。④疏肝法、补肾养心法的协同作用对记忆力调节作用较全面、稳定。更多还原

【Abstract】 Learning and memory decline belongs to "forgettery" "amnesia" in Traditional Chinese Medicine(TCM), and are the evidence of brain dysfunction. With the increase of stress in modern life, learning and memory decline has become one of the significant factor which affect people’s life. Memory decline can be caused by many factors, in young and middle-aged people, it is mainly caused by unhealthy lifestyle such as lack of exercises, staying up late, drink abuse, in which the focus is the memory decline caused by sleep-disorder and the sleep disorder could be imitated by sleep-deprivation. This study reconstructed animal model by sleep-deprivation to specify the neurobiology mechanism of Sini San’s effect on sleep-deprivation rats model learning and memory ability.Part I Theoretical ResearchBy searching on the relationship between sleep and learning and memory of doctors through the ages, find that the formation and consolidation of sleep and learning memory has a strong relation with liver, heart and kidney. In the process of learning and memory, liver, heart, kidney, heart can interrelated each other by qi and blood, meridians and five-zang shen, and they have a division in information processing. People’s cognition of the outside world starts from the heart, then form a preliminary impression by thinking and analysing, and judge that by recalling and storing the memory, and finally consolidate the memory in brain. The liver controlling conveyance and dispersion, the regulation of liver soul penetrate in the process, and intercourse between heart and kidney is also important for waxing and waning of yin-yang. As for the learning disorder caused by lack of sleeping, theoretically, the main differenciation types are not just limited to the regulation of heart and kidney. Liver loss dispersion on conveyance and dispersion, lack of nourisment of heart spirit and deficency of kidney essence can be involved.Part II Experimental studyExperiment 1 Sleep-deprivation rats and the evaluation of its learning and memory abilityObjective:Reconstruct learning and memory disorder model, evaluate the model by animal behavior and observe Jiajian Sini San’s effect on rats’learning and memory ability. Method: Reconduct learning and memory disorder rats model by sleep-deprivation, record the changes of general condition, weight, Y-maze score and SOD in serum of rats. Results:①changes of general conditions:the control group is normal from the third day of molding to the end, the model group and treatment group show mania, aggressivity, high alert, bite, unhairing, loose stool in different level, the condition of Sini San group and composition treatment group alleviate than model group and Shenghui Decoction group.②changes of weight:recording the weight changes of the 1,3,5,8 day from molding to the end, found that the weight of control group increased, the model group and other 3 treatment group decended in different level, compared to control group, the remaining four groups difference significant, but no difference among the four groups.③Y-maze scores:test the wrong reaction times and active avoidance response rate of the rats on the 0 and 7 day. On the 0 day, there is no difference among groups, on the 7 day, the wrong reaction times of model group and other three treatment groups are more than the control group, there are significant differences among model group, Sini San treatment group and Shenghui decoction treatment group, composition treatment group have significant difference; comparing to the model group, the active avoidence response rate of composition treatment group increases and has significant difference while the Sini San group has significant difference, too.④The expression of SOD in serum:comparing to the control group, the content of SOD in model group decrease and has significant difference; the content of SOD in Shenghui decoction treatment group reduced; whlie in Sini San group and composition group, there is no significant difference. Comparing to the model group, the content of SOD in Sini San group and composition group increase significantly, between both groups, the former group has a lower content of SOD than the composition group. Conclusion:①The rats’memory decline with general conditions and weight changes can be caused by sleep-deprivation.②The soothing liver, tonifying kidney and nourishing heart method can improve the memory decline caused by sleep-deprivation, the soothing liver method can improve part of the memory ability and tonifying kidney and nourishing heart method has no obvious effect.ExperimentⅡBehavioral changes of sleep-deprivation rats and the regulation of Jiajian Sini SanObjective:Observe the effect of Jiajian Sini San on the cognition of sleep-deprivated rats to a new enviroment. Method:use self-made desert field box to do open-field test. Results:on the 0 day of molding, the indexes of open-field test has no significant difference among the groups. On the 7 day, comparing to the control group, the upright number of model group decrease significantly, thus has significant difference; comparing to the model group, the unright number of the three treatment group increase and have significant difference. As for the grid-across number, comparing to the control group, the model group decrease significantly; comparing to the model group, Sini San group increase dramatically, and has significant difference, Shenghui decoction group increases significantly, thus has significant difference. On the 7 day of molding, comparing to the control group, the residence time in central region of the control group induces and has significant difference; and when comparing to the model group, the residence time of Shenghui decoction group and composition group are shorter and have significant difference, the residence time of Sini San group is shorter and has notable difference. As the decoration time, the compositon group is more than the model group, and there are no significant difference among other groups. Conclusion:①Sleep-deprivation can reduce the rats’space exploration ability, excitability and cognition ability, but no evident effect on adaption.②The soothing liver, tonifying kidney and nourishing heart method can improve the emotion and cognition disorder caused by sleep-deprivation, and they can regulate them from different respects.ExperimentⅢThe effect of sleep-deprivation on the gene and immune positive things expression of BDNF in rats’hippocampus and the receptor TrkB, and the regulation of Jiajian Sini San.Objective:Observe the effect of Jiajian Sini San on the expression of BDNF and TrkB related to learning and memory in sleep-deprivated rats. Method:Use immuohistochemistry and real-time PCR to detect the positive material and gene expression of BDNF and TrkB in hippocampal CA3 area and DG area. Conclusion:①Immune positive matter of BDNF can be seen in hippocampal CA3 and DG area, which is claybank granulate, oval or cone, and they distribute in cytoplasm and nucleus of CA3 area pyramidal cell and DG area granulocyte.②The protein expression of BDNF:the results of CA3 area shows the MOD of BDNF of model group and three treatment groups decrease comparing to the control group, the difference is significant; comparing to the model group, the MOD of composition group increases and has significant difference, the Sini San group and Shenghui decoction group has no significant difference. The DG area results show the MOD of BDNF in model group and other three treatment group decrease compared to the control group, the difference is significant; comparing to the model group, there is no significant difference among Sini San group, Shenghui decoction group and composition group.③Gene expression of BDNF:comparing to the control group, hippocampal BDNF mRNA of model group decrease, the difference is notable; and there is no significant difference between model group and Sini San group or Shenghui decoction group; but in composition group, the hippocampal BDNF mRNA expression increase dramatically, and has significant difference.④The protein expression of TrkB:The results from CA3 area show that TrkB MOD of model group and the other three treatment groups decrease compared to the control group, the difference is significant; comparing to the model group, the MOD of Sini San group and compostion group increase and has notable difference, and Sini San group has a lower expression than the composition group. The results from DG area show TrkB positive materials MOD of model group and the three treatment groups decrease compared to the control group, and the difference is notable; comparing to the model group, three treatment groups MOD have no significant difference.⑤Gene expression og TrkB:comparing to the control group, mRNA of TrkB in model group decreases and has significant difference; there is no significant difference among Sini San, Shenghui decoction and composition groups; comparing to the model group, the mRNA expression of TrkB in Sini San group and composition group increase remarkably, the difference is notable; the mRNA expression of TrkB in Shenghui decoction has no evident increase. Conclusion:①Sleep-deprivation can decrease protein and gene expression of hippocampal BDNF and TrkB.②The composition can increase the gene expression of hippocampal BDNF and TrkB.③The soothing liver regulatory point of is narrow relatively and the method can improve expression of TrkB.Experiment IV Gene expression of nonreceptor tyrosine kinase Fyn in sleep-deprived rats’hippocampus and the intervention of Jiajian Sini SanObjective:To observe the effect of Jiajian Sini San on Fyn gene expression in sleep-deprived rats’hippocampus, identify the relationship between memory improvement and Fyn, BDNF, TrkB. Method:The gene expression of Fyn will be detected by realtime fluorescence quantitative PCR. Results:compared to the control group, Fyn mRNA expression in model group hippocampus decreases remarkably with very significant difference; compared to the model group, Fyn mRNA expression in Shenghui decoction group hippocampus increases, and has significant difference, the compostion Fyn mRNA increases significantly with notable difference, when there is no significant difference in Sini San group. Conclusion:①Sleep-deprivation can decrease the gene expression of Fyn in hippocampus.②Tonifying kidney and nourishing heart method and composition can improve the sleep-deprivation induced learning and memory decline by regulating the gene expression of Fyn, especially the composition group.③The gene expression of Fyn, BDNF and its receptor in sleep-deprivation rats’ hipocampus has consistency, and the regulatory function on learning and memory decline of the three protein may share the same signal path.Experiment Y The protein expression of ERK and pERK in. sleep-deprivated rats’ hippocampus and the regulatory function of Sini San.Objective:To observe the effect of Jiajian Sini San on ERK and pERK expression in sleep-deprivated rats’hippocampus, and identify the relationship between the effect and nonreceptor tyrosine kinase and brain-derived neurotrophic factors. Method:Observe the expression of ERK and its homocystein thiolactone by using western blot. Results:①The protein expression of ERK2:comparing to the control group, the expression of ERK2 with Shenghui and composition group increase; in model group and Sinisan group,there is no difference. comparing to the model group, the expression of ERK2 with Shenghui and composition group increase with siginificant difference.②The protein expression of pERK2: comparing to the control group, the pERK2 in composition groups improve while expression of the model group induces; comparing to the model group Shenghui and composition group increase.Conclusion:①Sleep-deprivation can significantly decrease the protein expression of ERK2 and pERK2 which are related to memory-formation.②The composition and tonifying kidney and nourishing heart method can improve ERK2 and pERK2 decline in sleep-deprivated rats’hippocampus, more obvious in pERK2 expression.③ERK2 and its Homocystein thiolactone, together with BDNF and Fyn, may participate in learning and memory decline caused by sleep-deprivation.SummaryThis study is based on sleep theory, five-zang spirit theory, to explore the mechanism of sleep formation and learning memory from liver, heart and kidney. People’s cognition of the outside world starts from the heart, then make a preliminary impression by thinking and analysing, then to judge that by recalling and storing the memory, and finally solid the memory in brain. The liver controlling conveyance and dispersion, the regulation of liver soul penetrate in memory formation and consolidation. Five-zang spirit, especially the liver, heart and kidney are of key importance to sleep-induced learning and memory disorder. By analysing the experimental results, we make conclusions as followings:①Deprive sleep from rats for 7 days continuously can cause learning and memory impairment.②The composition of Sini San and Shenghui decoction can improve the sleep-deprivation-induced learning and memory impairment by regulating the signalling transduction pathways ERK-pERK/BDNF /TrkB/Fyn in hippocampus.③The effect target of Sini San and Shenghui decoction is narrow, they improve memory ability from behavior, some proteins and genes.④The joint action of soothing liver and tonifying kidney, nourishing heart on memory improvement is compre-hensive and stable.更多还原