疏肝补肾养心法对疲劳所致学习记忆力下降作用的机理研究

【作者】 马佳美；

【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】 随着社会的发展,人们生活节奏加快,学习及工作压力也在无形中增大,疲劳因此成为现代人的所要面对的共同问题。疲劳包含体力疲劳及脑力疲劳,表现为四肢倦怠无力、情绪异常、反应能力及学习记忆力下降等。而记忆的能力又会影响学习成绩及工作效率,本课题以此为研究方向,由中医整体辨证思维探讨疲劳与记忆的关系,探索改善疲劳状态从而提高记忆力的方法。由于体力及脑力疲劳是并存且相互影响的,所以我们从疲劳及记忆力下降对心肝肾的病理表现影响之理论研究着手,通过实验研究,对疲劳所致学习记忆改变的中医证候学特点及其分子生物学机制进行探讨,以疏肝及补肾养,法同时着手治疗,改善由疲劳引发的学习记忆下降。因此更好地发挥中医整体观治病特点,并为改善记忆力下降奠定基础。1理论研究“疲劳”在古代以“懈怠”、“解惰”、“四肢倦怠”等来记载类似的症状,属于“劳倦”范畴,中医古籍中其实并没有分为心理性疲劳(脑力疲劳)或生理性疲劳(体力疲劳)。疲劳是由于大量的体力、脑力耗损而造成机体在生理及心理不同层面的功能失调。过度的劳力及劳神是相互影响的,并且累及心、肝、肾三脏,使之正常生理表现无法发挥,如肝失疏泄、肾精不充等,从而影响精、气、血、神。传统医学中学习记忆的功能是归属于“神志”的范畴,而“神”分属五脏所掌管。心藏神,主宰一切心理活动。通过认知与五神脏理论依据提示,由“肝魂”提取“肾志”所储存的长期记忆,并将“脾意”的决策信息一同传送至“心”,说明心、肝、肾三脏在学习记忆的认知过程中的重要性。因此本课题以肝、肾、心、脑之间的关系,从疲劳影响学习记忆的下降病理机制为基础,提出通过疏肝以及补肾养心法治疗改善疲劳状态的理论,理论包括阻断学习记忆能力下降的发生和直接提高学习记忆的能力的两方面内容。2实验研究本课题实验研究部分,首先以衰减全反射-傅里叶变换红外光谱(ATR-FTIR)技术对实验所选用方药进行检测,以此实验进行药物质量监控,与文献比对后证实实验用药具有药品可信度,并且推测其中所含的多醣类及氨基类物质与神经传导有关。动物实验采用复合疲劳模型以模拟更接近现实状况的疲劳病理状态,并由行为学实验证明疲劳影响学习记忆的功能。突触可塑性(Synaptic plasticity)被认为是产生学习和记忆的神经化学基础,主要由不同的蛋白激酶和蛋白磷酸化的活性特点,通过磷酸化对突触蛋白进行调控。据此,本研究以神经颗粒素(Neurigranin, Ng).蛋白激酶C(Protein kinaseC, PKC)及Ca2+-CaM依赖性蛋白激酶(Ca2+/Calmodulin-Dependent Protein KinaseⅡ. CaMKⅡ)等三个与突触可塑性密切相关的指标作为研究对象,应用Western Blot与Real-time PCR实验技术观察三者在疲劳大鼠海马CAl区的蛋白质及mRNA表达的变化,以及使用疏肝、补肾养心的三组方药干预后的改变情形。以此分析疲劳所致学习记忆下降的机理和中医心肝肾间的关系。ATR-FTIR检测实验用药组方及个别单味药将四逆散、生慧汤之组方、单味药以及四逆散与生慧汤合方以衰减全反射-傅里叶变换红外光谱(ATR-FTIR)技术进行检测,实验通过与文献比对研究中所用的配方颗粒,结果经分析后实验用药具有可信度。以单味药、复方交叉比对,结果显示四逆散及生慧汤皆有较明显的多醣类及氨基的特殊峰。疲劳大鼠动物模型的建立及评价本实验结合大鼠游泳运动及睡眠剥夺的复合方法建立疲劳动物模型,用药组给予疏肝的四逆散,补肾养心的生慧汤以及兼具调补心肝肾作用的四逆散及生慧汤合方进行干预。由一般生理指标,如毛色、体重等及血清LDH作为判定疲劳的指标。证实本实验成功的建立疲劳模型,并且三个方药都具一定的改善疲劳作用。疲劳大鼠学习记忆能力的评定本研究通过Y迷宫实验,观察正确反应率、错误反应次数、达标所需训练次数和总反应时间,藉以评价疲劳大鼠的学习记忆能力,以及各方药的调节作用。Y迷宫结果显示,造模前各组大鼠的学习记忆能力无显着差异。造模后,模型组与对照组比较,正确反应率明显降低(p<0.01),错误次数明显增多(p<0.01),达到学会所需次数明显增多(p<0.01)。说明疲劳可能导致大鼠学习记忆能力的下降,疏肝组、补肾养心组以及合方组与模型组相比,均能提高正确反应率,减少错误反应次数及达标所需次数。说明疏肝、补肾养心及合方组均能改善疲劳大鼠学习记忆能力的下降,其中以合方组改善实验动物的学习记忆能力最为明显。疲劳大鼠记忆力下降与Ng、PKC及CaMKⅡ表达之关系行为学实验结束后,各组随机选取大鼠6只,取其左侧半脑进行Western Blot实验,右侧半脑进行Real time PCR实验,检测Ng、PKC及CaMKⅡ在疲劳大鼠海马CA1区中蛋白质及mRNA的表达水平。Ng为脑特异性蛋白质,是PKC的作用底物,亦是Ca2+敏感性钙调蛋白(calmodulin,CaM)的结合蛋白,通过调控Ca2+和CaMKⅡ,参与学习记忆过程。结果显示,模型组的疲劳大鼠海马CA1区Ng蛋白质及mRNA的表达水平较对照组下降,由于Ng的表达与学习记忆能力成正相关,而推测疲劳可导致学习记忆受损。通过疏肝的四逆散、补肾养心的生慧汤以及兼补心肝肾的合方的调节,实验显示,其Ng的蛋白质及mRNA表达皆较模型组提升。三个用药组中Ng的表达上升趋势以合方最为显效,其次为补肾养心方,再其次为疏肝方。另外,在PCR实验中显示,合方之提升作用明显超越对照组,说明合方效用优于单纯的疏肝或补肾养心作用。另外,合方对Ng的提升作用不论在蛋白质水平或基因水平中都与疏肝组有差异性。基于PKC的蛋白质和mRNA水平在疲劳大鼠海马CA1区的表达,模型组较对照组显着降低,说明经由睡眠剥夺及游泳的复合模型降低了PKC水平,可能造成海马区神经细胞的受损。与模型组相比,三个用药组对PKC的蛋白质及基因水平都具有明显的上调作用。其中在Western blot实验中更可见补肾养心组及合方组上调作用明显高于疏肝组。虽然Western blot实验中合方组与补肾养心组并无显着差异,且在PCR实验中的差异更小,但以其中的趋势看的出疏肝补肾养心合方用药组是优于另两组,而补肾养心组又优于疏肝组。因此,疏肝及补肾养心的合方虽然没有明显的加成作用,但对于PKC的表达仍显示其具有较好的上调结果。在检测Thr286磷酸化CaMKⅡ的Western blot实验中,结果显示模型组显着低于对照组。PCR实验中模型组CaMKⅡ的基因表达也有极为显着的下调。由于CaMKⅡThr286自身磷酸化为诱导LTP的形成之所须,说明模型组可能因造模期间海马损伤而影响其CaMKⅡ的基因及蛋白表达,进而使CaMKⅡ在Thr286位点的磷酸化现象也受影响。三个用药组对于CaMKⅡ的调节于实验结果中可见：蛋白质水平中,疏肝组及合方组与模型组相比对Thr286磷酸化的CaMKⅡ的上调作用非常显着；补肾养心组则略微上调,但不具差异性。就基因水平而论与模型组相比,合方组具有上调作用,单纯的疏肝及补肾养心组则效用不具差异性。并且,三个用药组的CaMKⅡ基因表达水平与对照组相比是显着低于对照组,说明用药可能不作用于基因水平上,而在蛋白质水平上也有可能在影响CaMKⅡ的磷酸化途径上,进而使Thr286磷酸化的CaMKⅡ的上调,其机制还有待进一步研究。3小结本课题对疏肝法与补肾养心法改善疲劳所致学习记忆力下降的作用情况进行了研究。理论研究从中医对心、肝、肾与疲劳影响学习记忆的关系入手,认为疲劳是由体力及脑力疲劳相互作用,且与心、肝、肾之关系密切,因此由疏肝法和补肾养心法都能改善疲劳后的学习记忆能力下降的问题。实验通过行为学实验,及检测分析参与学习记忆机制中的Ng、PKC和CaMKⅡ的蛋白质和基因水平表达,观察发现合方方药对三者的表达皆有一定程度的上调作用,说明藉由疏肝及补肾养心可以有效改善因疲劳而造成的学习记忆能力下降。更多还原

【Abstract】 As society develops, the accelerating pace of life has increased pressure among everyone in working and studying. Fatigue has become a common problem to modern people; it includes physical fatigue and mental fatigue, which shows symptoms of tiredness, mood disorder, slow responses, and decline of learning and memory ability. As memory effects on learning performance and work efficiency, our main research goal is to study the relationship between fatigue and memory under the concept of Traditional Chinese Medicine, and also to search for methods to improve memory loss. Since the coexistence of physical and mental fatigue and affect between each other, our study underlies the theory of the effect on heart, liver and kidney pathology, which led by fatigue and memory loss, and combine experimental researches to have a better perspective on the mechanism of how soothing liver and tonifying kidney with nourishing heart can improve memory loss induced by fatigue.1. Theory ResearchThere was no difference between physical or mental fatigue in ancient Chinese medicine. Symptoms were described as "slack", "laziness", "limb fatigue", which all refers as "fatigue". With great amount of physical and mental wear out will cause physical and psychological dysfunction. Excessive physical fatigue and mental fatigue will interact, and cause damage to the normal physiological manifestations on liver and kidney, showing symptoms such as stagnation in liver Qi or lack of kidney essence, therefore essence, Qi, blood and the spirit will be affected. In TCM theory, learning and memory are being categorized in "Mind". The five spirits are in charged by the five viscera. Heart masters "God", and takes charge of all mental activities. In the five spirit cognitive theory, "liver soul" extracts the long-term memory in "kidney will" and delivers the message to "heart" along with the decision information made in "spleen intent". This implies the key role of heart, liver and kidney in the recognition process.Therefore, according to the relationship between liver, kidney, heart and brain. and the connection with the pathological mechanism of fatigue induced memory loss, we propose the theory of recover fatigue to block the occurrence of learning and memory ability decline by soothing liver and tonifying kidney with nourishing heart, and also to improve memory loss directly by soothing liver and tonifying kidney with nourishing heart. 2. Experimental ResearchAt first, we applied ATR-FTIR (Attenuated Total reflection-Fourier transform infrared spectroscopy) technique to exam the herbs we use. In fatigue animal experiment, combine swimming and multiple platform method in order to imitate the realistic pathological state of fatigue. And in behavior experiment, it has been proven that memory loss can be induced by fatigue. Learning and memory is underling synaptic plasticity in neurology, and is activated by different protein kinase activity and regulate by protein phosphorylation on synapse. Therefore our research focus on the study of Neurogranin (Ng), Protein Kinase C (PK.C) and Ca2+-CaM-dependent protein kinase (CaMKⅡ). application of Western Blot and Real-time PCR technique to observe the level of protein and mRNA in the Hippocampal CA1 region of fatigue rats. And the expression difference after applying soothing liver and tonifying kidney with nourishing heart formulas.ATR-FTIRBy using ATR-FTIR technique screen all of the single herbs in Si Ni San and Sheng Hui Tang. Experimental herbs were proven to be reliable. And we speculate polysaccharide and amino substance are contained in these formulas, as also relevant to signal transmit in neurons.Combined fatigue animal modelCombine swimming and multiple platform sleep deprivation methods as composite fatigue animal model on sleep deprivation rats, which is an ideal method to avoid additional stress, inflammation and other occurred error. Animal model is proved by animal’s weight, coat color and other general observations, tests of CK and LDH in serum, and other common indicators of fatigue testing. Use soothing liver Qi formula:Si Ni San, and tonify kidney and nourishing heart formula:Sheng Hui Tang has also proved to be effective to reduce fatigue and improve memory.Evaluation of learning/memory in fatigue ratsEvaluate learning/memory changes through Y maze. Results show, comparing model group and normal group, the correct response rate was significantly lower (p< 0.01), numbers of error increased (p< 0.01); numbers of time to learn have increased (p< 0.01). Comparing soothing liver group, tonify kidney and nourishing heart group, combined formula group and model group have all increased correct response rate (p< 0.01), decreased in error rate (p< 0.01). Indicate that comparing with model group, soothing liver group, tonify kidney and nourishing heart group and combined formula group can elevate the correct respond rate and decline error rate. The combined formula group has the most significant learning ability among all.The expression of Ng, PKC, CaMKⅡin the Hippocampal CA1 region of fatigue ratsAfter the behavioral experiment,6 rats were randomly selected in each group, obtain the left hemisphere of the brain for Western Blot test, the right half for Real time PCR experiments, examine the protein and mRNA expression levels of Ng, PKC and CaMKⅡin the Hippocampal CA1 region of fatigue rats.Ng is the brain-specific protein, is the substrate of PKC, and is also the binding protein of calmodulin. which is involved in the process of learning and memory by regulating Ca2+ and CaMKⅡ. Results show, both Ng protein and mRNA expressions in the Hippocampal CA1 region of fatigue rats decrease in the model group than in control group. According to the expression of Ng was positively related to learning and memory, we speculate that fatigue can reduce learning and memory ability. Comparing to model group the Ng protein and mRNA expression level increased after intake soothing liver and tonifying kidney plus nourishing heart formulas. Among the three treatment groups, the combined formula has the most effective influence, followed by tonifying kidney and nourishing heart formula, and then soothing liver formula. Indicates combining soothing liver and tonifying kidney and nourishing heart has the most effective influence than methods only applying soothing liver or tonifying kidney and nourishing heart.The level of PKC protein and mRNA expression in the Hippocampal CA1 region of fatigue rats, decrease significantly in the model group than in control group. Base on the decrease level of PKC. might indicate damage occurred to nerve cells in the hippocampus. Both PKC protein and mRNA expression level increased after intake soothing liver and tonifying kidney formulas. In the result of Western blot, tonify kidney group and combined lormula group perform better than soothing liver group. Although, the results of western blot and PCR in combined formula group are just slightly higher than tonify kidney and nourishing heart group, but they indicate, with combining soothing liver and tonifying kidney with nourishing heart has the most effective influence.The detection of Thr286 phosphorylation CaMKⅡin Western blot experiments showed that the model group was significantly lower than the control group, results are the same based on gene expression level. CaMKⅡThr286 autophosphorylation is cruial to induce LTP, thus indicates there might be damge in hippocampus while animal model experiment, lead to affect CaMKⅡexperession. In three treatment groups. the Thr286 phosphorylation of CaMKⅡin soothing liver group and in combine formula group were significant upregulation comparing to model group. And there’s only slightly upregluate in tonifing kidney and nourishing heart group, but there are not difference. On the gene level, the combined formula group has significant upregulation on CaMKⅡcomparing with the model group. But there are no difference with soothing liver group and tonifying kidney and nourishing heart group. Plus, the regulation comaring between the protein and gene espression are different, maybe because the formulas don’t effect on CaMKⅡgenes, and directly effect on CaMKⅡphosphorylation.ConclutionOur project focus on the mechanism of the regulation of memory loss induced by fatigue with the methods of using soothing liver and tonifying kidney. Our theory is to use heart, liver and kidney as a breakthrough to regulate memory loss lead by fatigue, according to the impact between fatigue and memory loss, and the connections within heart, liver and kidney. With the experimental research, by ATR-FTIR. behavioral experiment, and by Western blot and PCR technique examing the expression of Ng, PKC and CaMKⅡ, we discover that the regulation of soothing liver and tonifying kidney and nourishing heart formulas can improve memory loss induced by fatigue. Thus, the method of soothing liver and tonifying kidney with nourishing heart can regulate memory loss by recovering fatigue or to improve memory directly.更多还原