The Study Of Compound Formulae Of Qingre Lidan Jiedu On Learning And Memory Ability Of Rat Model With Copper-Load Based On Synaptic Plasticity And MAPK/ERK Signaling Pathway

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.3Results3.1Compared 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).4Conclusion4.1This 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.