| As traditional medicine,Traditional Chinese medicine maintains the health of the Chinese nation and provides hope for the treatment of alzheimer's disease.Azlheimer's diesas(AD)is a progressive neurodegenerative disease,the typical characteristic neuropathologic changes of AD are extracellular neuroinflammatory plaques(SP)and intracellular neurofibrillary tangle(NFT).As the main component of SP,Amyloid precursor protein(APP)is produced successively by hydrolysis of?-secretase and?-secretase.However,most amount of the APP are metabolized and released soluble and blocked by?-secretase.Inhibiting the generation of APP and reducing the deposition of A?is the key to the prevention and treatment of AD.Objective:In this study,we take APP/PS1transgenic AD mice as the animal model and observe the effect of CC granule on the learning and memory behavior of APP/PS1transgenic AD mice,then observe the changes in the structure and morphology of neurons in the brain tissue of APP/PS1 transgenic AD miceon the basis of the behavioral.Detect the effect of CC granule to the levels of related secretases which plays an important tole on the formation of A?through the decomposition of the APP,find out some of ways and links related with A?aggregation pathwaythe which CC granule targeted.Observe the effect of CC granule to neural plasticity,elucidated the inhibiting mechanism of CC granule on APP?A?protein expression.Also,detect the differential biomarkers in the serum of APP/PS1 transgenic AD mice to find out metabolic pathways related to the prevention and treatment of AD by compound compound granule,provide more scientific evidences for the development of CC granule on the treatment of AD.Methods:100 three months old APP/PS1 transgenic mice were randomly divided into 5 groups:APP/PS1transgenic AD model group,Donepezil(0.92 mg/kg)treatment group,CC granule low dose(2.0g/kg)?medium dose(4.0g/kg)and high dose(8.0g/kg)treatment group,20 mice in each group.Another 20 three-month-old C57BL/6J mice were chosen as normal control group.All administration groups received relevant medicine for successive 6 months.After 6 months drug administration,the changes on the learning and memory behaviors were investigated by Morris water maze test and step-down phases test;then the animals were sacrificed,brain tissue was taken out and fixed,the pathomorphism in hippocampus CA1 zone was detected by HE staining,Congo red staining and Modified Bielschowsky's staining methods;the changes of myelin sheath,microtubule,and microfilament in myelinated nerve of hippocampus CA1 zone were detected by electron microscope;immuno histochemistry and Western blot methods were used to test APP?PS1,BECA?A??GAP43?Doublecortin and tau protein expression in the brain tissue.The endogenous metabolites in the serum were analyzed using NMR in conjunction with multivariate and statistical techniques.Results:(1)MWM experimental results showed that,compared to normal group,the number of times of entering escape platform and effective area in AD model group mice was significantly reduced(P<0.01),the movement time of effective area and swimming distance in effective area were significantly reduced(P<0.01).Compared to APP/PS1 transgenic AD model group mice,the number of platform entrance time and effective area entrance time,movement time of effective area and swimming distance in effective area of Donepezil treatment group,CC granule high dose group and CC granule medium doses group treatment mice were significantly increased(P<0.01);the number of platform entrance time and effective area entrance time of CC granule low dose treatment mice were also increased(P<0.05),but no any significant difference on movement time of effective area and swimming distance of effective area.(2)The results of the step-down phases experiment showed that,compared with the normal control group,the response period of APP/PS1 transgenic AD model mice was significantly prolonged(P<0.01),latency time was significantly shortened(P<0.01),the number of errors was significantly increased(P<0.01).Compared with APP/PS1 transgenic AD model group,the response period of Donepezil treatment group,CC granule low dose?medium dose and high dose treatment group mice has been shortened(P<0.01?0.05),latency time was significantly prolonged(P<0.01?0.05),the number of errors was significantly reduced(P<0.01?0.05).(3)Hippocampus HE staining results show that,hippocampus cell layer of the CC granule treatment group Hippocampal cells layer is complete,the arrangement is in order,compact structure,and normal morphology,distribution is balanced,the intracellular structure was normal,the staining showed that the cytoplasm of the cytoplasm was light blue,the cytoplasm of the protuberant cytoplasm was light red,the nucleolus was large and obvious,the cytoplasm was abundant,there were a small number of retracted necrotic neurons,the nuclear membrane was clear,and a small number of glial cells were visible,and no changes of glial cells were observed.Congo red staining observation result showed large number of proliferated glial cells in the CC granule low dose treatment APP/PS1 transgenic AD model group,forming senile plaques with a large number,surrounding the blue cell band,the cells were small and sparsely arranged.Bielschowsky's modified staining result showed there were some nerve fibers were thickened,misaligned and dense into clumps in the CC granule low dose treatment APP/PS1 transgenic AD model group,the deep-dyed trailing tail shape inside the axon went deep into the cell protuberances,forming a filamentous helical twist of nerve fibers tangles in the cytoplasm.(4)The number of APP?PS-1?A??BACE and tau positive cells in the hippocampal CA1 region of the APP/PS1 transgenic AD model group were increased and the cytoplasm is colored deeply,the mean number and average density of APP?PS-1and BACE positive cells were significantly higher than those of normal control group?CC granule medium dose and high dose treatment group(P<0.01?0.05),but no significant difference with Donepezil(0.92 mg/kg)treatment group and CC granule low dose(2.0g/kg)treatment group(P>0.05);Compared with the normal control group,the average number and average density of A?40-42positive cells in the hippocampal CA1 of APP/PS1 transgenic AD model group were significantly increased(P<0.05).Compared with APP/PS1 transgenic AD model group,the average number and average density of A?40-42positive cells in the hippocampal CA1 of CC granule medium dose and high dose treatment group decreased significantly(P<0.01),the average number and average density of A?40-42positive cells in the hippocampal CA1 of Donepezil treatment group and CC granule low dose also decreased significantly(P<0.05);the average number and average density of tau protein positive cells in the hippocampal CA1 of APP/PS1transgenic AD model group were similar to the normal control group,the average number and average density of tau protein positive cells in the hippocampal CA1 of Donepezil treatment group,CC granule low dose?medium dose and high dose(8.0g/kg)treatment group were decreased compared to APP/PS1 transgenic AD model group(P<0.05?0.01);Compared with APP/PS1 transgenic AD model group,the average density of Doublecortin positive cells in the hippocampal CA1 of normal control group?Donepezil treatment group?CC granule medium dose and high dose treatment group were significantly increased(P<0.05?0.01),but no significant difference with CC granule low dose(2.0g/kg)treatment group(P>0.05);the average number and average density of GAP43 positive cells in the hippocampal CA1 of APP/PS1 transgenic AD model group were significantly decreased compared with normal control group?CC granule low dose?medium dose and high dose treatment group(P<0.05?0.01),but no significant difference compaired with Donepezil treatment group(P>0.05).(5)Western blot results showed that compared with the normal control group,the bands of APP,PS1,BECA,A?and tau protein of APP/PS1 transgenic AD model were significantly thicker and darker,indicating increased protein expression.Compared with the APP/PS1 transgenic AD model group,the APP,PS1,BECA,A?and tau protein of Donepezil treatment group,CC granule low dose?medium dose and high dose treatment group were significantly thinner and lighter in bands,indicating decreased protein expression.Western blot results of GAP43 and Doublecortin showed that the bands from APP/PS1 transgenic AD model group were lighter than normal control group and treatment groups,indicating decreased protein expression.(6)The results of NMR metabonomics test showed that,compared with the normal control group,the concentration of the serum of APP/PS1 transgenic AD model group increased and the concentration of glycine?tyrosine?phenylalanine?1-methylhistidine?methionine?malonic acid?lactic acid statistically decreased(P<0.05);the concentration of lactic acid?alanine and formic acid were increased;the concentrations of isoleucine?tyrosine?phenylalanine?doxin-glucose?acetic acid?methionine and creatine were decreased in the serum of CC granule low dose treatment group(P<0.05);levels of isoleucine?tyrosine?phenylalanine??-glucose?acetate and carnitine decreased in the serum of CC granule medium dose treatment group(P<0.05);the concentrations of lactic acid?alanine?puerarin and glucomannan were increased and the concentrations of1-methylhistidine were decreased in the serum of CC granule high dose treatment group(P<0.05);The serum concentrations of lactic acid?tyrosine and glutar-glucose were increased?while the concentrations of isoleucine?alanine?hydroxybutyric acid?1-methyl-histidine and carnitine were decreased in the Donepezil treatment group(P<0.05).Compared with the APP/PS1 transgenic AD model group,the concentrations of lactic acid?glycine?tyrosine and pyruvate were increased in the serum of CC granule low dose treatment group(P<0.05);the concentration of lactic acid and pyruvate were increased and the concentrations of alanine were decreased in the serum of CC granule medium dose treatment group(P<0.05);the concentration of lactic acid was increased and the concentration of unsaturated lipid was decreased in the serum of mice in the serum of CC granule high dose treatment group(P<0.05);the concentrations of lactic acid?glycine and pyruvate were increased and the content of 1-methylhistidine was decreased in the serum of the Donepezil treatment group(P<0.05).Conclusion:(1)The cognitive function of the transgenic AD model of APP/SP1 was impaired,which was reflected in reduced learning ability,poor spatial positioning ability and memory loss.CC granule could be affective to the enhancement of cognitive function of APP/SP1 transgenic AD mice,it can improve the learning and memory ability,as well as spatial search ability of APP/SP1 transgenic AD mice.(2)APP/SP1 transgenic AD model mice showed decreased number of nerve cells in the hippocampal CA1 region,part of the cells were unclear in contour,the cell structure was severely damaged,the cell nucleus morphology was irregular,and the mitochondria and synapses were irregular.Treatment with CC granule can improve the structure of mitochondria and synapses in the hippocampal CA1 region of APP/SP1 transgenic AD mice.This function is closely related to its improvement of learning and memory ability of APP/SP1 transgenic AD mice.(3)The expression levels of APP,PS-1,A?,tau and BACE proteins in the hippocampal CA1 region of APP/SP1transgenic AD model mice were significantly increased,which was one of the key factor to causes cognitive dysfunction in APP/SP1 transgenic AD model mice,however 6months treatment with compound treatment could effectively reduce the expression of APP,PS-1,A?,tau and BACE proteins in the APP/SP1 transgenic AD model mice,reduce the deposition of A?,protect the brain tissue nerve cells against to toxicity and damage caused by A?,also reduce the expression of tau protein,reduce the excessive phosphorylation of tau protein inhibit the formation of neuronal fiber tangles,reduce the formation of old age plaques,then prevent the occurrence and development of AD.(4)6months treatment with CC granula could significantly increase the expression of GAP43and Doublecortin proteins in the hippocampal,and improve neural plasticity,alleviate axon injury,promote the regeneration of neurons and axon,promote the recovery of brain function,accelerate the improvement of cognitive function of APP/SP1 transgenic AD mice.(5)Glycine?tyrosine?phenylalanine?lactic acid?glucose-glucose and glucose-glucose can be used as the differential bio-markers in the serum of APP/SP1transgenic AD model mice.After 6 months of administration of CC granule,the content of tyrosine in the serum of APP/PS1 transgenic AD mice was significantly increased,suggesting that CC granula administration could accelerate the synthesis of DA by increasing the content of tyrosine,It inhibits the formation and aggregation of A?,reduces the toxicity of A?,protects the nerve cells and improves the cognitive function of APP/PS1 transgenic AD mice,as well as improves the learning and memory ability.This may be another mechanism of action of CC granula in the prevention and treatment of Alzheimer's disease.(6)The CC granule can significantly improve the cognitive function of the APP/SP1 transgenic AD model mice and high dose of CC granule works better,and there is a dose-dependent relationship between the efficacy and the dose. |
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