基于突触可塑性和MAPK/ERK信号通路的清热利胆解毒方对铜负荷大鼠学习记忆行为的干预机制研究
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摘要
1.研究背景及目的
肝豆状核变性（Wilson disease,WD）是一种常染色体隐性遗传的铜代谢障碍疾病，临床上WD引起神经系统变性可使整个神经系统受累，以基底节、小脑系统神经损伤为基础的锥体外系症状包括以双上肢为主的震颤，言语表达能力下降、含糊不清，肌张力增高、双膝关节痛、行走障碍，吞咽障碍及表现为孤僻、欣快和记忆力下降等神经精神系统症状。
学习记忆的实质是信号的传导和处理的过程，这一切功能的产生都是以特定结构的形成为其基础。行为依赖性的突触可塑性正是学习记忆形成的机制，后天训练获得了空间辨别性学习记忆功能后在海马内会引起突触数量的增多、突触活性区膜面积的增加、突触小泡数量和体积的增加等一系列形态学变化，这些变化被认为是空间辨别性学习记忆在海马结构内引起了突触形态学的可塑性变化。
MAPK通路是细胞外信号引起核反应的细胞信息传递的汇聚通路之一，MAPK主要由三个途径组成，即细胞外信号调节激酶（ERK）、c-jun末端调节激酶和蛋白调节激酶p38。其中ERK包括ERK1和ERK2，大量研究表明ERK与长时程增强（LTP）存在效应关系，阻止了ERK的激活，也
同时阻止海马CA1区LTP的诱导，而LTP和学习、记忆过程密切相关，被许多学者命名为“学习、记忆的突触模型”，从而确定了ERK与学习记忆的关系。ERK激活后还可以使海马细胞的树突发生形态学改变，说明ERK可能参与了神经元形态学可塑性的形成过程，有助于信息的传递和接受。MAPK/ERK信号转导通路与学习、记忆密切相关，细胞内多条途径可激活ERK，活化的ERK从多方面参与学习、记忆的形成过程。
前期对WD患者的研究发现，以神经症状为主的脑型WD患者存在有严重的记忆障碍，而其他临床表型的WD患者记忆商也偏低。WD患者智能、记忆损害程度与疾病病程的演变过程相一致,提示铜造成的神经细胞损伤对WD患者的学习记忆功能产生了一定的影响。导师所在课题组在临床实践中证明:以清热利胆解毒方为主的中西医结合治疗可以使WD患者智商总体水平有不同程度的提高，尤其可使视空间技能显著提高，体现在观察能力、长时间视觉记忆能力、远见性、计划性和结构综合判断能力均增强。
本实验主要开展以下研究：建立铜负荷大鼠模型，观察清热利胆解毒方中药对铜负荷大鼠学习记忆行为及MAPK/ERK信号通路表达的影响，运用电子显微镜技术检测清热利胆解毒方中药对铜负荷大鼠海马区微结构的影响，初步探讨清热利胆解毒方中药治疗WD学习记忆功能障碍的机制。
2方法
2.1实验分为5组：分别为空白对照组，模型对照组，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组；
2.2按照文献复制铜负荷大鼠模型；
2.3使用清热利胆解毒方中药对各治疗组铜负荷大鼠进行干预，选用青霉胺为阳性对照药物；
2.4Morris水迷宫法检测铜负荷大鼠学习记忆行为；
2.5免疫组化法检测MAPK/ERK蛋白的表达；
2.6电镜技术观察铜负荷大鼠大鼠海马区微结构变化；
3.结果
3.1与正常组相比，模型对照组，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组大鼠均不同程度的表现出进食减少、活动量减小，皮毛光泽度差，部分有毛发脱落等。随着治疗进行，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组大鼠的一般状况均有所改善。
3.2Morris水迷宫行为学实验：定位航行实验中模型组大鼠的平均潜伏期较空白组明显延长(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药组及青霉胺组较模型组平均潜伏期显著减少(P﹤0.05)，组间比较无显著差异。空间探索试验中模型组大鼠的穿越平台较空白组明显减少(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药组及青霉胺组较模型组穿越平台显著增加(P﹤0.05)，组间比较无显著差异。
3.3免疫组化法检测MAPK/ERK信号通路表达：MAPK/ERK蛋白阳性信号呈棕黄色，模型组及各治疗组可见阳性细胞表达，模型组阳性细胞数表达较空白组显著减少(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药及青霉胺组较模型组MAPK/ERK阳性细胞数表达显著增加(P﹤0.01)，组间比较无显著差异。
3.4电镜观察海马区突触可塑性变化：采用透射电镜技术，观察各组大鼠海马CA3区神经元突触界面超微结构的变化，模型组与空白对照组比较，模型组大鼠神经元突触间隙明显变宽(P﹤0.05)，PSD厚度明显变薄(P﹤0.01)。与模型组比较，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药及青霉胺组较模型组突触间隙明显变窄(P﹤0.05)，PSD厚度明显增加(P﹤0.05)。
4.结论
4.1本研究采用铜负荷方法建立了铜过量负荷大鼠模型，模型组可见学习记忆功能的变化，该模型可在一定程度上反应铜过量沉积导致神经系统损伤引起学习记忆功能变化的的病理机理。
4.2本研究发现铜过量负荷大鼠模型可见学习记忆功能的下降、清热利胆解毒方中药对铜负荷大鼠有改善学习记忆功能的作用。
4.3本研究发现铜过量负荷大鼠模型有MAPK/ERK信号通路蛋白的阳性表达减少，各治疗组较模型组明显改善。提示：铜过量负荷后大鼠脑组织MAPK/ERK信号通路蛋白的阳性表达减少可能是导致学习记忆功能损害的原因之一，清热利胆解毒方中药可能通过对MAPK/ERK信号通路的表达增加而发挥改善学习记忆功能的作用。
4.4本研究发现铜过量负荷大鼠模型有神经元突触界面超微结构的变化，各治疗组突触超微结构较模型组明显改善。提示：铜过量负荷后大鼠脑组织神经元突触界面超微结构改变可能是导致学习记忆功能损害的原因之一，清热利胆解毒方中药可能通过对通过调节神经元突触界面超微结构来发挥改善学习记忆功能的作用。
1Objectives:
Wilson’s disease (WD) is an autosomal recessive disorder of coppermetabolism caused by decreased biliary excretion. Clinically, Widespreadcopper accumulation in central nervous system (CNS) can make thedegeneration of CNS. Patients with WD exhibit a broad range of symptoms,including the extrapyramidal symptoms that based on basal ganglia andcerebellum damage present with both upper extremities tremor, decline inverbalization, hypermyotonia, knee-joint pain, walking disorders, deglutitiondisorders, and performance as the autism, euphoria, memory decline and otherneuropsychiatric symptoms.
The essence of learning and memory is the process of signal transductionand processing. Generation of all of this functionality is based on theformation of its specific structure. Behavior-dependent synaptic plasticity isthe mechanism of learning and memory formation. It will cause increases inthe number of synapses, the membrane area of the synaptic active zone, thenumber and volume of synaptic vesicles and other series of morphologicalchanges in the hippocampus when the function of spatial discriminativelearning and memory being obtained by training, whcih are considered that the spatial discriminative learning and memory in the hippocampus causesmorphological synaptic plasticity changes.
The MAPK pathway is one of the cell information disseminationaggregation pathway of the nuclear reaction caused by extracellular signal.MAPK is composed of three main ways, namely the extracellularsignal-regulated kinase (ERK), the end of the c-jun kinase of p38-regulatedkinase and protein regulation. ERK includes ERK1and ERK2. Studies haveshown that the effect relationship exists between the ERK and long-termpotentiation (LTP), which blocks ERK activation, and also prevents theinduction of LTP in hippocampal CA1region. LTP is closely related to theprocess of learning and memory, which is named “synaptic model of learningand memory” by many scholars. Thus it determines the relationship betweenthe ERK and learning and memory. ERK activation can also make themorphological changes of dendrites in hippocampal cells, indicating that ERKmay be involved in the establishment of neuron morphological plasticity,which is contribute to the delivery and reception of the information. MAPK/ERK signal transduction pathway is closely related to the learning andmemory and ERK activation caused by the intracellular pathway is involved inthe establishment of learning and memory from the aspects.
The preliminary study found that WD patients with mainly neurologicalsymptoms have severe dysmnesia and other clinical phenotypes in patientswith low memory quotient. The degrees of intelligence and memoryimpairment in WD patients are consistent with the evolution of the diseasecourse, indicating that the nerve cells damage caused by the copper has acertain impact on learning and memory function in WD patients. The theresearch group of tutor in clinical practice proved: Compound Formulae ofQingre Lidan Jiedu based on combination of Chinese traditional and Westernmedicine can improve the IQ level of WD patients in different degrees, and particularly can significantly improve the visuospatial skills reflected in theability to observe, long term visual memory ability, vision, plan and structurecomprehensive judgment.
This study was carried out the following research: the establishment ofthe copper load rat model, the observation of the impact of CompoundFormulae of Qingre Lidan Jiedu on learning and memory behavior and MAPK/ERK signaling pathway expression in the copper load rat, the effect ofCompound Formulae of Qingre Lidan Jiedu on micro-structure of thehippocampus in the copper load rat by electron microscopy detection andinitially discussing the mechanism of learning and memory dysfunction inWD by Compound Formulae of Qingre Lidan Jiedu treatment.
2Methods：
2.1Rats were randomly were divided into5groups: blank control group,model control group, Compound Formulae of Qingre Lidan Jiedu group,Compound Formulae of Qingre Lidan Jiedu plus penicillamine group,penicillamine group.
2.2Establishment of the copper load rat model according to the literature.
2.3Employ the Compound Formulae of Qingre Lidan Jiedu intervention in thetreatment group copper loaded rats, and select penicillamine as positivecontrol drug.
2.4Detect the learning and memory behavior of copper loaded rats by Morriswater.
2.5Detect MAPK/ERK protein expression by immunohistochemical method.
2.6Observe the micro-structure changes of the hippocampus in copper loadedrats by electron microscopy.
3Results
3.1Compared with the blank control group, model control group, CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre Lidan Jiedu plus penicillamine group and penicillamine group rats showed thateating less, Less activities, the fur gloss got worse and hair loss. With thetreatment, the general condition of Compound Formulae of Qingre LidanJiedu group, Compound Formulae of Qingre Lidan Jiedu plus penicillaminegroup and penicillamine group rats improved in different degrees.
3.2.Morris water maze experiment: the average incubation period of the modelrats was significantly longer in the navigation test compared with the blankcontrol group (P <0.01); the average incubation period of CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre LidanJiedu plus penicillamine group and penicillamine group rats significantlydecreased compared with the model group (P <0.05) and no significantdifferences existed between groups. The through platform number of themodel rats significantly decreased in space exploration test compared with theblank control group (P <0.01); the through platform number of CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre LidanJiedu plus penicillamine group and penicillamine group rats significantlyincreased compared with the model group (P <0.05) and no significantdifferences existed between groups.
3.3MAPK/ERK protein expression by immunohistochemical method:positive signal of MAPK/ERK protein appeared brownish yellow; there werepositive cells inthe model group and the treatment group; the number of positive cells in themodel group significantly decreased compared with the blank control group (P<0.01); the number of positive cells in the Compound Formulae of QingreLidan Jiedu group, Compound Formulae of Qingre Lidan Jiedu pluspenicillamine group and penicillamine group significantly increased comparedwith the model group (P <0.05) and no significant differences existed betweengroups.
3.4Synaptic plasticity changes by electron microscopy in the hippocampus: toobserve the ultrastructural changes of synaptic interface in hippocampal CA3neurons by transmission electron microscopy; the synaptic cleft in the modelgroup significantly got wider compared with the blank control group (P <0.05)and the PSD thickness significantly got thinner (P <0.05); the synaptic cleft inthe Compound Formulae of Qingre Lidan Jiedu group, Compound Formulaeof Qingre Lidan Jiedu plus penicillamine group and penicillamine groupsignificantly narrowed compared with the model group (P <0.05) and the PSDthickness significantly increased (P <0.05).
4Conclusion
4.1This study used the the copper load method to establish the excessivecopper load rat model; there were visible changes in learning and memoryfunction in the model group; the model reflected the pathomechanism of thechanges in learning and memory function caused by copper excess depositionleading to nervous system damage in some degrees.
4.2This study found that the decline in learning and memory function existedin copper load rat model and Compound Formulae of Qingre Lidan Jiedu canimprove the learning and memory function of copper load rat.
4.3Our study found MAPK/ERK signaling pathway protein expressiondecreased in copper load rat model, and each treatment group had somesignificant improvements compared with the model group, suggesting thatMAPK/ERK signaling pathway protein expression in brain after copperexcess deposition may be one of the reasons of learning and memorydysfunction, and Compound Formulae of Qingre Lidan Jiedu may play a rolein improving learning and memory function through increasing expression ofMAPK/ERK signaling pathway.
4.4Our study found there were neuron ultrastructural changes of synapticinterface in copper load rat model, and each treatment group had some significant improvements compared with the model group, suggesting that theultrastructural changes of synaptic interface in brain neurons after copperexcess deposition may be one of the reasons of learning and memorydysfunction, and Compound Formulae of Qingre Lidan Jiedu may play a rolein improving learning and memory function through adjusting the neuronultrastructure of synaptic interface.
肝豆状核变性（Wilson disease,WD）是一种常染色体隐性遗传的铜代谢障碍疾病，临床上WD引起神经系统变性可使整个神经系统受累，以基底节、小脑系统神经损伤为基础的锥体外系症状包括以双上肢为主的震颤，言语表达能力下降、含糊不清，肌张力增高、双膝关节痛、行走障碍，吞咽障碍及表现为孤僻、欣快和记忆力下降等神经精神系统症状。
学习记忆的实质是信号的传导和处理的过程，这一切功能的产生都是以特定结构的形成为其基础。行为依赖性的突触可塑性正是学习记忆形成的机制，后天训练获得了空间辨别性学习记忆功能后在海马内会引起突触数量的增多、突触活性区膜面积的增加、突触小泡数量和体积的增加等一系列形态学变化，这些变化被认为是空间辨别性学习记忆在海马结构内引起了突触形态学的可塑性变化。
MAPK通路是细胞外信号引起核反应的细胞信息传递的汇聚通路之一，MAPK主要由三个途径组成，即细胞外信号调节激酶（ERK）、c-jun末端调节激酶和蛋白调节激酶p38。其中ERK包括ERK1和ERK2，大量研究表明ERK与长时程增强（LTP）存在效应关系，阻止了ERK的激活，也
同时阻止海马CA1区LTP的诱导，而LTP和学习、记忆过程密切相关，被许多学者命名为“学习、记忆的突触模型”，从而确定了ERK与学习记忆的关系。ERK激活后还可以使海马细胞的树突发生形态学改变，说明ERK可能参与了神经元形态学可塑性的形成过程，有助于信息的传递和接受。MAPK/ERK信号转导通路与学习、记忆密切相关，细胞内多条途径可激活ERK，活化的ERK从多方面参与学习、记忆的形成过程。
前期对WD患者的研究发现，以神经症状为主的脑型WD患者存在有严重的记忆障碍，而其他临床表型的WD患者记忆商也偏低。WD患者智能、记忆损害程度与疾病病程的演变过程相一致,提示铜造成的神经细胞损伤对WD患者的学习记忆功能产生了一定的影响。导师所在课题组在临床实践中证明:以清热利胆解毒方为主的中西医结合治疗可以使WD患者智商总体水平有不同程度的提高，尤其可使视空间技能显著提高，体现在观察能力、长时间视觉记忆能力、远见性、计划性和结构综合判断能力均增强。
本实验主要开展以下研究：建立铜负荷大鼠模型，观察清热利胆解毒方中药对铜负荷大鼠学习记忆行为及MAPK/ERK信号通路表达的影响，运用电子显微镜技术检测清热利胆解毒方中药对铜负荷大鼠海马区微结构的影响，初步探讨清热利胆解毒方中药治疗WD学习记忆功能障碍的机制。
2方法
2.1实验分为5组：分别为空白对照组，模型对照组，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组；
2.2按照文献复制铜负荷大鼠模型；
2.3使用清热利胆解毒方中药对各治疗组铜负荷大鼠进行干预，选用青霉胺为阳性对照药物；
2.4Morris水迷宫法检测铜负荷大鼠学习记忆行为；
2.5免疫组化法检测MAPK/ERK蛋白的表达；
2.6电镜技术观察铜负荷大鼠大鼠海马区微结构变化；
3.结果
3.1与正常组相比，模型对照组，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组大鼠均不同程度的表现出进食减少、活动量减小，皮毛光泽度差，部分有毛发脱落等。随着治疗进行，清热利胆解毒方组，清热利胆解毒方组加青霉胺组，青霉胺组大鼠的一般状况均有所改善。
3.2Morris水迷宫行为学实验：定位航行实验中模型组大鼠的平均潜伏期较空白组明显延长(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药组及青霉胺组较模型组平均潜伏期显著减少(P﹤0.05)，组间比较无显著差异。空间探索试验中模型组大鼠的穿越平台较空白组明显减少(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药组及青霉胺组较模型组穿越平台显著增加(P﹤0.05)，组间比较无显著差异。
3.3免疫组化法检测MAPK/ERK信号通路表达：MAPK/ERK蛋白阳性信号呈棕黄色，模型组及各治疗组可见阳性细胞表达，模型组阳性细胞数表达较空白组显著减少(P﹤0.01)，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药及青霉胺组较模型组MAPK/ERK阳性细胞数表达显著增加(P﹤0.01)，组间比较无显著差异。
3.4电镜观察海马区突触可塑性变化：采用透射电镜技术，观察各组大鼠海马CA3区神经元突触界面超微结构的变化，模型组与空白对照组比较，模型组大鼠神经元突触间隙明显变宽(P﹤0.05)，PSD厚度明显变薄(P﹤0.01)。与模型组比较，青霉胺加清热利胆解毒方中药组、清热利胆解毒方中药及青霉胺组较模型组突触间隙明显变窄(P﹤0.05)，PSD厚度明显增加(P﹤0.05)。
4.结论
4.1本研究采用铜负荷方法建立了铜过量负荷大鼠模型，模型组可见学习记忆功能的变化，该模型可在一定程度上反应铜过量沉积导致神经系统损伤引起学习记忆功能变化的的病理机理。
4.2本研究发现铜过量负荷大鼠模型可见学习记忆功能的下降、清热利胆解毒方中药对铜负荷大鼠有改善学习记忆功能的作用。
4.3本研究发现铜过量负荷大鼠模型有MAPK/ERK信号通路蛋白的阳性表达减少，各治疗组较模型组明显改善。提示：铜过量负荷后大鼠脑组织MAPK/ERK信号通路蛋白的阳性表达减少可能是导致学习记忆功能损害的原因之一，清热利胆解毒方中药可能通过对MAPK/ERK信号通路的表达增加而发挥改善学习记忆功能的作用。
4.4本研究发现铜过量负荷大鼠模型有神经元突触界面超微结构的变化，各治疗组突触超微结构较模型组明显改善。提示：铜过量负荷后大鼠脑组织神经元突触界面超微结构改变可能是导致学习记忆功能损害的原因之一，清热利胆解毒方中药可能通过对通过调节神经元突触界面超微结构来发挥改善学习记忆功能的作用。
1Objectives:
Wilson’s disease (WD) is an autosomal recessive disorder of coppermetabolism caused by decreased biliary excretion. Clinically, Widespreadcopper accumulation in central nervous system (CNS) can make thedegeneration of CNS. Patients with WD exhibit a broad range of symptoms,including the extrapyramidal symptoms that based on basal ganglia andcerebellum damage present with both upper extremities tremor, decline inverbalization, hypermyotonia, knee-joint pain, walking disorders, deglutitiondisorders, and performance as the autism, euphoria, memory decline and otherneuropsychiatric symptoms.
The essence of learning and memory is the process of signal transductionand processing. Generation of all of this functionality is based on theformation of its specific structure. Behavior-dependent synaptic plasticity isthe mechanism of learning and memory formation. It will cause increases inthe number of synapses, the membrane area of the synaptic active zone, thenumber and volume of synaptic vesicles and other series of morphologicalchanges in the hippocampus when the function of spatial discriminativelearning and memory being obtained by training, whcih are considered that the spatial discriminative learning and memory in the hippocampus causesmorphological synaptic plasticity changes.
The MAPK pathway is one of the cell information disseminationaggregation pathway of the nuclear reaction caused by extracellular signal.MAPK is composed of three main ways, namely the extracellularsignal-regulated kinase (ERK), the end of the c-jun kinase of p38-regulatedkinase and protein regulation. ERK includes ERK1and ERK2. Studies haveshown that the effect relationship exists between the ERK and long-termpotentiation (LTP), which blocks ERK activation, and also prevents theinduction of LTP in hippocampal CA1region. LTP is closely related to theprocess of learning and memory, which is named “synaptic model of learningand memory” by many scholars. Thus it determines the relationship betweenthe ERK and learning and memory. ERK activation can also make themorphological changes of dendrites in hippocampal cells, indicating that ERKmay be involved in the establishment of neuron morphological plasticity,which is contribute to the delivery and reception of the information. MAPK/ERK signal transduction pathway is closely related to the learning andmemory and ERK activation caused by the intracellular pathway is involved inthe establishment of learning and memory from the aspects.
The preliminary study found that WD patients with mainly neurologicalsymptoms have severe dysmnesia and other clinical phenotypes in patientswith low memory quotient. The degrees of intelligence and memoryimpairment in WD patients are consistent with the evolution of the diseasecourse, indicating that the nerve cells damage caused by the copper has acertain impact on learning and memory function in WD patients. The theresearch group of tutor in clinical practice proved: Compound Formulae ofQingre Lidan Jiedu based on combination of Chinese traditional and Westernmedicine can improve the IQ level of WD patients in different degrees, and particularly can significantly improve the visuospatial skills reflected in theability to observe, long term visual memory ability, vision, plan and structurecomprehensive judgment.
This study was carried out the following research: the establishment ofthe copper load rat model, the observation of the impact of CompoundFormulae of Qingre Lidan Jiedu on learning and memory behavior and MAPK/ERK signaling pathway expression in the copper load rat, the effect ofCompound Formulae of Qingre Lidan Jiedu on micro-structure of thehippocampus in the copper load rat by electron microscopy detection andinitially discussing the mechanism of learning and memory dysfunction inWD by Compound Formulae of Qingre Lidan Jiedu treatment.
2Methods：
2.1Rats were randomly were divided into5groups: blank control group,model control group, Compound Formulae of Qingre Lidan Jiedu group,Compound Formulae of Qingre Lidan Jiedu plus penicillamine group,penicillamine group.
2.2Establishment of the copper load rat model according to the literature.
2.3Employ the Compound Formulae of Qingre Lidan Jiedu intervention in thetreatment group copper loaded rats, and select penicillamine as positivecontrol drug.
2.4Detect the learning and memory behavior of copper loaded rats by Morriswater.
2.5Detect MAPK/ERK protein expression by immunohistochemical method.
2.6Observe the micro-structure changes of the hippocampus in copper loadedrats by electron microscopy.
3Results
3.1Compared with the blank control group, model control group, CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre Lidan Jiedu plus penicillamine group and penicillamine group rats showed thateating less, Less activities, the fur gloss got worse and hair loss. With thetreatment, the general condition of Compound Formulae of Qingre LidanJiedu group, Compound Formulae of Qingre Lidan Jiedu plus penicillaminegroup and penicillamine group rats improved in different degrees.
3.2.Morris water maze experiment: the average incubation period of the modelrats was significantly longer in the navigation test compared with the blankcontrol group (P <0.01); the average incubation period of CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre LidanJiedu plus penicillamine group and penicillamine group rats significantlydecreased compared with the model group (P <0.05) and no significantdifferences existed between groups. The through platform number of themodel rats significantly decreased in space exploration test compared with theblank control group (P <0.01); the through platform number of CompoundFormulae of Qingre Lidan Jiedu group, Compound Formulae of Qingre LidanJiedu plus penicillamine group and penicillamine group rats significantlyincreased compared with the model group (P <0.05) and no significantdifferences existed between groups.
3.3MAPK/ERK protein expression by immunohistochemical method:positive signal of MAPK/ERK protein appeared brownish yellow; there werepositive cells inthe model group and the treatment group; the number of positive cells in themodel group significantly decreased compared with the blank control group (P<0.01); the number of positive cells in the Compound Formulae of QingreLidan Jiedu group, Compound Formulae of Qingre Lidan Jiedu pluspenicillamine group and penicillamine group significantly increased comparedwith the model group (P <0.05) and no significant differences existed betweengroups.
3.4Synaptic plasticity changes by electron microscopy in the hippocampus: toobserve the ultrastructural changes of synaptic interface in hippocampal CA3neurons by transmission electron microscopy; the synaptic cleft in the modelgroup significantly got wider compared with the blank control group (P <0.05)and the PSD thickness significantly got thinner (P <0.05); the synaptic cleft inthe Compound Formulae of Qingre Lidan Jiedu group, Compound Formulaeof Qingre Lidan Jiedu plus penicillamine group and penicillamine groupsignificantly narrowed compared with the model group (P <0.05) and the PSDthickness significantly increased (P <0.05).
4Conclusion
4.1This study used the the copper load method to establish the excessivecopper load rat model; there were visible changes in learning and memoryfunction in the model group; the model reflected the pathomechanism of thechanges in learning and memory function caused by copper excess depositionleading to nervous system damage in some degrees.
4.2This study found that the decline in learning and memory function existedin copper load rat model and Compound Formulae of Qingre Lidan Jiedu canimprove the learning and memory function of copper load rat.
4.3Our study found MAPK/ERK signaling pathway protein expressiondecreased in copper load rat model, and each treatment group had somesignificant improvements compared with the model group, suggesting thatMAPK/ERK signaling pathway protein expression in brain after copperexcess deposition may be one of the reasons of learning and memorydysfunction, and Compound Formulae of Qingre Lidan Jiedu may play a rolein improving learning and memory function through increasing expression ofMAPK/ERK signaling pathway.
4.4Our study found there were neuron ultrastructural changes of synapticinterface in copper load rat model, and each treatment group had some significant improvements compared with the model group, suggesting that theultrastructural changes of synaptic interface in brain neurons after copperexcess deposition may be one of the reasons of learning and memorydysfunction, and Compound Formulae of Qingre Lidan Jiedu may play a rolein improving learning and memory function through adjusting the neuronultrastructure of synaptic interface.
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