| ObjectiveTo explore the etiological factor and effect of replenishingkidney-essence and remove phlegm therapy on treating Alzheimer'sdisease(AD) in theory. And to establish a novel complex rat model ofAlzheimer's disease(AD) which was induced by injection of basalnucleus of Meynert (BNM) with ibotenic acid(IBO) and 25-35β-amyloid peptide(Aβ25-35). And to observe the effects of replenishingkidney-essence and removing phlegm therapy on learning memory, theactivity of cholinergic system,pathological observation and theubiquitin-protea-some passway in the hippocampus and cerebral cortexfor analyzing the mechanism of action of the therapy for AD.Methods1. The normal 15 months old Wistar rats (n=60) were randomlydivided into normal group, Aβ25-35 model group, IBO model group,andAβ25-35+IBO model group, There were fifteen rats in every group.theninduced by injection of BNM with IBO, accumulatio Aβ25-35 and thecomposition of IBO and Aβ25-35 to establish AD animal modelrespectively. The rat water-maze test and the pathological observation ofthe hippocampus and cerebral cortex were carried out to evaluate themouse model of AD.2. The normal 15 month old Wistar rats(n=70) were randomlydivided into seven groups:normal group,blank group,AD model group,Huperzine A-Zhulin Antun treating group,high-dose,middle-dose andlow-dose of BCHT groups.There were ten rats in every group.To induceAD, five groups rats received injection of IBO andβ-amyloid peptide(Aβ) with BNM through stereospecific instrument. After treated for fourweeks, Three doses of BuShen HuaTan (BSHT) decoction wereadministered by oral garage. Huperzine A-Zhulin Antun tablets servedas control drug. Normal group , model group and blank group wereadministered by saline water.After treated for four weeks,Morris water maze was used toobserving the ability of space learning and memory.To observe neuro-fibrillary tangles (NFT) of brain tissue through silver staining, senileplaque(SP) through methanol Congo red, ultramicro-pathological ofhippocampus were observed by transmission electron microscope(TEM),radioimmunoassay was used to assaying the activity of acetylcholine(Ach), acetylcholinesterase(AchE) and choline acetyl transferase(ChAT),immunohistochemistry and reverse transcription-polymerase chainreaction (RT-PCR) methods were used to detecting the content ofubiquitin protein (Ub) in hippocampus and cerebral cortex. Western blotand Enzyme Linked-Immuno-Sorbent Assay(ELISA) were used todetecting volume dose of ubiquitin-activating enzyme(E1) protein,ubiquitin-con-jugating enzymes EPF(E2-EPF)and ubiquitin carboxyterminal hydrolase (UCH-L1) in hippocampus and cerebral cortexhomogenate.To observe their effects on the above marks ,we analyze thetherapeutic mechanism of BSHT decoction in experimental AD modelrats.Results1. In the behavior experiment,the ability of learning gain and thecompetence of information reserve of the composite model groupshowed the significant differences which were compared with the simplex factor model group(P＜0.01).Furthermore,the result of patholo-gical observation revealed that the number of neurofibrillary tangles andβ-amyloid protein plaque in the hippocampus increased and thedegeneration of hippocampal and cerebral cortex neurons in compositemodel group rats.2. In the behavior experiment, average incubation period ofhigh-dose, middle-dose and low-dose groups have obvious shortencompared to model group (p＜0.01); Percentage of quadrant ofhigh-dose, middle-dose and low-dose groups have obvious increasedcompared with model group (p＜0.01), 20％and 40％area have obviousdecreased compared with model group(p＜0.01), in space research ofmodel group, the frequency in span of platform localization andpercentage of area have obviously decerased compared to normal andblank group(p＜0.01). However, high-dose,middle-dose and low-dosegroups have obviously increased,compared with model groups(p＜0.01,), and have dose-effect relationship.3. The result of electron and light microscope have approved thatthe volume of neuron of hippocampus decreased and denaturated inmodel group and replenishing kidney-essence, removing phlegm therapycan recovery those pathological change.4. The activity of Ach and ChAT in hippocampus cortical area waslower in AD model rats than that of other group's rats obviously(p＜0.01), but the activity of AchE climbed sharply. There is nosignificant difference between the bland and normal group (p＞0.05); thesignificant differences were observed between the model group and thehigh-,middle-,low-dose group (p＜0.01).There is no difference between high-treated group and middle- dose group(p＞0.05).5.The level of these proteins of ubiquitin-proteasome pathway haveno obvious difference between normal group and blank group(p＞0.05).the expression of Ub and E2-EPF in model group have markedincreased than normal group(p＜0.01, p＜0.05), but the expression of E1and PG9.5 in model group have marked decreased than normal group(p＜0.01, p＜0.05), After administrated by BSHT decoction, the expres-sion of Ub,E2-EPF,E1 and PG9.5 have remarkedly increased,especiallythe high-dose group.Moreover, the high-,middle-dose and low-dosegroups have dose-effect relationship (p＜0.01, p＜0.05).Conclusion1. Kidney-essence weak and phlegm stasis is the basic etiologicalfactor for AD,and replenishing kidney-essence, removing phlegmtherapy is operative on treating AD.2. The Animal Model of Alzheimer's disease induced by compositeAβ25-35 and IBO injected into basal nucleus of Meynert of old ratsimulated some characteristics of human AD to someextent,whichmaybe used as a novel model to study AD and its drug treatment.3.replenishing kidney-essence,removing phlegm therapy canimprove disturbance learning and momory, increase the activity of Achand ChAT, decrease the activity of AchE,to repair the function ofCholinergic system, and improve pathological morphous change ofhippocampus brain tissue in AD model rats.4. replenishing kidney-essence and removing phlegm therapy canincrease the level of Ub, E1, E2-EPF and PGP9.5, enhance the activity ofubiquitin-proteasome pathway.
|
PDF Full Text Request