The Neuron Damage Induced By β-amyloid And The Protective Effect Of Donepezil For Hippocampal Formation

PrefaceAlzheimer's disease(AD) is a kind of degenerative nerve diseases that clinically characterized by progressive dementia.With the gradual ageing of the population,the morbidity of AD increases year by year.It has brought heavy burden to the society and families,and has become one of the fatal diseases that hazard to human health.AD is pathologically characterized by senile plaques(SP) formed by pathological deposition ofβ-amyloid(Aβ),neurofibrillary tangles(NFTs) and neuron loss.By now,the pathogenesy of AD is not clear,but more and more evidences suggest that neuron apoptosis is the important factor to induce neuron loss,a great quantity of neuron loss result in AD's cognition function degression.It is considered that cells' apoptosis is the result of a series of cysteine proteinases cascade reaction event,caspase-3 is a downstream protein of the cascade reaction,and it is the real executor for apoptosis and can cause cell death through degradating substrates of intracellular.Hippocampal formation correlates with learning and retention closely,furthermore,it is the earliest and easiest part of damaged in AD.So the neuron apoptosis in hippocampal formation directly affect AD's cognition.Now,a majority of scholars consider that the deposition of Aβin brain tissues is the common path of various factors to cause AD,which is also the key factor of AD to form and develope.At present,some research indicate that Aβanomaly deposition being able to initiate series cascade reaction,induce nervous toxicity,injure nervous system,to lead patients to appear cognition dysfunction.But which enzymes have participated in Aβguiding nerve toxicity,and these molecules interaction and regulation mechanism have failed to elucidate.Therefore,further study of the mechanism that Aβdestroys nerves have considerable theory and clinical significance. Phosphatidylinositol 3-kinase/Akt(PI3-K/Akt)signaling is one of the important intracellular signal pathway of promoting cell survival.Akt is a downstream protein kinase of PI3-K,which is activated through phosphorylation after the PI3-K is activated by extracellular signals,such as growth factor receptors.Activated Akt can phosphorylate apoptosis related genes,to promoted apoptosis genes inactivated,while anti-apoptosis genes activated,thus,inhibit neuron apoptosis and promote neuron survival.Then whether the neuron damage induced by Aβis concerned with inhibiting Akt phosphorylation? the studies about this are few now.Donepezil is a kind of selective cholinesterase inhibitors,commonly cure AD. Most clinical experiments confirm that donepezil can obviously improve patients' learning and retention functions.Now,studies found that DN may reduce free radicals creation and restrain excitability toxicity of glumatic acid,but whether it can restrain the neuron apoptosis induced by Aβand inhibit the expression of caspase-3,as well as, whether those effects have relation with activated Akt? The reports about these questions are few.In this experiment,we cultured PC12 cells in vitro and made AD rat model through injecting Aβinto lateral cerebral ventricle and meanwhile to give donepezil treatment,as well as their mechanisms to detect Aβ's neurotoxicity and the neuron protective effect of donepezil.So as to provide experiment according for clinical treatment,and the pharmaprojects about prevent or treat AD provide theory according.Materials and MethodsIn vitro shudy1.Cell culture:PC12 cells were cultured in RPMI-1640 medium with 10%fetal bovine serum and 5%horse serum,serial subcultivation in 37℃,5%CO₂ incubator.2.MTT assay:adopt different concentration of aggregated Aβ_25-35 induce PC12 cells in vitro cultured,detect cells' survival rate,elect suitable concentration of Aβ_25-35 to made AD damaged neural cell model.The cells were grouped into control group and model group,normal PC12 cells as control group,Aβintervention group as model group.3.Flow cytometry assay:detect above tow groups PC12 cells apoptosis ratio.4.Immunohistochemistry:detect above tow groups PC12 cells staining for caspase-3 expression.In vivo study1.Experimental animal and grouping:36 male Wistar rats of three month, weighing 250g～300g were provided by Animal Experimental Center of China Medical University.After raising 1 week,the rats were randomly to divide into control group,model group and treat group,with 12 in each.The rats in model and treat group were treated with injecting 10μl condensed Aβ_25-35 into lateral cerebral ventricle,the rat in control group were injected same dose physiological saline in the same method.In the 3d after made model,the treat group were start to give donepezil(1.5mg·kg^-1·d^-1,ip), the rat in control and model group were treated with same dose physiological saline,the process continues for 4 weeks.2.Morris water maze for testing learning and remembrance ability of every group rats,and record the average escape latency in every segment.3.Immunohistochemical staining for caspase-3 expression in rat hippocampal formation in every group.4.Western blot for T-Akt,P-Akt expression in rat hippocampal formation.Statistics analysisImage collection analysis system was applied.The datas were expressed as Mean±SD and were analyzed by SPSS 11.5 statistical package.The differences among group were compared with one-way analysis of variance.p＜0.05 is regarded as the significant difference and p＜0.01 is regarded as the extremely significant difference.ResultsThe results of in vitro experiment:1.Results of MTT assay:The PC12 cells activity is degression on dose dependent, the best concentration of Aβ_25-35 is 20μM.2.Results of Flow cytometry:the apoptosis ratio of PC12 cells in the model group is increased obviously compared with control group(p＜0.01).3.Results of immunohistochemistry:The positive reaction product of caspase-3 in model group was stained obviously increased compared with control group(p＜0.01). The results of in vivo experiment:1.Results of Morris water maze:All segmental escape latency in model group were prolonged compared with control group(p＜0.01).The escape latency in treat group were shortened compared with model group(p＜0.01).2.Results of immunohistochemistry:The positive reaction product of caspase-3 was stained buffy or brown,most of them deposited in pyramidal cell layer of hippocampal CA1 and CA3,and granular cell layer of dentate gyrus.Compared with control group,the optical density of caspase-3 positive reaction product in model group was increased(p＜0.04).The optical density of caspase-3 positive reaction product in treat group was decreased compared with model group(p＜0.05).3.Results of western blot:The expression of T-Akt in hippocampal formation of three groups without significant difference.But the expression of P-Akt in model group is down regulation compared with control group(p＜0.05),while which in the treat group is up-regulation(p＜0.05).Conclusions1.Aβ_25-35 neurotoxicity partly through inducing neuron apoptosis,which possible concerned with promote the expression of caspase-3.2.Aβ_25-35 promote the expression of caspase-3 may concerned with the down reg ulation of Akt phosphorylation.3.The neuronprective effect of Donepezil related with inhibit the expression of caspase-3,which concerned with the up-regulation of Akt phosphorylation.