Mechanism Of Improvement Of Learning And Memory Performance By Total Salvianolic Acid In APPswe/PS1dE9 Mice

Alzheimer's disease(AD)is a common chronic progressive neurode-generative disease in elderly individuals characterized by memory disorder,acalculia,disorientation,aphasis,poor faculty of understanding and judgement,emotional and behavior disorders and so on.These symptoms have strong impact on the daily life of patients and family members.Most of AD were in sporadic form,however,familial form is less.The familial AD is an autosomal dominant genetic disease involved in point mutations in the gene of APP as well as PS-1,PS-2 and ApoE4 allelic genes.The main pathological changes of Alzheimer's disease are characterized by the presence of neuritic plaques,neurofibrillary tangles,loss of neurons,synaptic loss,granular vacuolar changes,as well as amyloid cerebrovascular changes.The theory of “A? cascade” is accepted at present involved in pathogenesis of AD.The anomalous deposition of A? peptide leads to mitochondrial dysfunction,oxidative damage and promoted hyperphosphorylation of tau protein and induces cell apoptosis eventually.A? is produced by beta-secretase and gamma-secretase from amyloid-precursor protein.A?consists of soluble fragments and insoluble fragments which result in cell destruction.The mutations in APP gene as well as PS-1,PS-2 result in increasing of insoluble fragments which activate A? cascade reaction.In addition,the pathogenesis of AD also includes immunologic dysfunction which consists of excessive activation of microglia and astrocyte and the excess inflammatory cytokines in some regions of the brain.The activated microglia function in antigen presentation and phagocytosis including in mediate inflammatory reactions and thus cause neuron damage.APPswe/PS1dE9(APP/PS1)transgenic mouse models have been developed in 1996.These specific transgenic mice harbor chimeric mouse/human familial AD genes for amyloid precursor protein(APP)and a mutant human presenilin 1(PS1).APP/PS1 transgenic mouse models mimic the formation of A? plaques within neural tissue and upregulation of microglia.The APP/PS1 transgenic mouse models are especially important for understanding disease etiology and testing new therapeutic procedures.Metabolomics plays an outstanding role in biomarker discovery and identification during the progressive course of AD,For example,cerebral glucose uptake in transgenic AD mice and patients is notably reduced,often before the typical changes occur.There are many kinds of AD mutant mice model at present,however,oxidative damage is greater in APP/PS1 double transgenic mice than in other mutant mice model.APP/PS1 mouse model is more suitable in evaluating the effect of antioxidant drugs.Total Salvianolic Acids(TSA),a type of polyphenolic compound,are extracted from the water-soluble effective parts in salvia miltiorrhiza bunge.TSA have been reported to play a role in clearing free radicals and have exact antioxidant effects in previous studies.In the present study,we used APP/PS1 transgenic mice to clarify whether TSA could induce neuroprotective effects on learning and memory.Furthermore,we discussed the mechanism of improvement of learning and memory performance by14-week-TSA treatment in APP/PS1 mice from the aspect of metabolism,AD-like pathology,glial-mediated neuroin?amma-tion and so on.Part one Behavior research on amelioration of learning and memory impairments in APPswe/PS1dE9 mice treated with TotalSalvianolic AcidObjective:To investigate whether treatment with TSA ameliorated learning and memory impairment in APP/PS1 mice by behavioral tests.Methods:1 A total of 30 male APP/PS1 mice were randomly divided into 3 groups.10 male littermate were treated as WT control.The APP/PS1 group and the WT control group were treated with an equal volume of vehicle(0.4ml/d NS,i.p.).The TSA-treated groups were treated with total salvianolic acid(Tianjintasly co.,LTD,Tianjin,China)at doses of 30 mg/kg·d and 60 mg/kg·d(TSA,i.p.),respectively.2 Daily treatment was administered to the mice from the age of 3.5months and had been kept for 14 weeks.The body weights were measured every week to adjust the administration dosage per mouse.3 After consecutive treatment for 14 weeks,behavioral research experiments including the NOR,MWM and step-through tasks on the mice were performed.The preference index(PI),average escape latency,frequency of passing across the location of platform,the time ratio for sailing inside the target quadrant and the latency in the step-through task were recorded to evaluate the learning and memory capability in four groups.During the period of the behavioral experiments,no treatment was administered to the mice in four groups.Results:1 The preference index(PI)of the APP/PS1 mice was decreased compared with that of WT control mice one hour after the acquisition memory phase in the task of NOR(P<0.01).Compared with the APP/PS1 trasgenic group,PI of the APP/PS1 mice increased after TSA treatment(30 mg/kg TSA,P<0.01;60 mg/kg TSA,P<0.05).In addition,the results demonstrated that it had no impact on the memory of the APP/PS1 mice 24 hours after acquisition memory phase [F(3,20)=0.611,P=0.612].2 In the spatial navigation trials of Morris water maze task,there was no significant difference in the escape latency of the four groups during the former three days(P>0.05).On the forth and the fifth day of the spatial navigation trials,the average escape latency in the APP/PS1 mice was prolonged appreciably compared with the WT control mice(P<0.01).Compared with APP/PS1 transgenic group,both doses of TSA treatment(i.p.)reversed the impairments in escape latency [(Day4: 30 mg/kg TSA,P<0.01;60 mg/kg TSA,P<0.05);(Day5: 30 mg/kg TSA,P<0.01;60 mg/kg TSA,P<0.01)],and improved the swimming strategy with which the mice tracked down the platform.In the probe trial,the latency of the APP/PS1 mice was substantially prolonged compared with that of the WT control mice(P<0.01).The latency decreased in APP/PS1 mice after TSA treatment(P<0.01)and there was no significant difference in the latency between the TSA-treated groups and the WT control group in the probe trial(P>0.05).The frequency of passing across the location of the target platform decreased in the APP/PS1 group versus the WT control group(P<0.05).Treatment with TSA multiplied the frequency crossing in the removal target platform relative to APP/PS1 group(30 mg/kg TSA,P<0.01;60 mg/kg TSA,P<0.05).The time ratio for sailing inside the target quadrant in APP/PS1 transgenic group decreased compared with the WT control group(P<0.01)and TSA treatment reversed the decrease compare with APP/PS1 group(30 mg/kg TSA,P<0.05;60 mg/kg TSA,P<0.01).3 Fear memory was influenced in four groups(x2=12.223,P=0.007).It was shown in step-through task that the escape latency reduced in APP/PS1 group compared with the WT control group(P<0.01).TSA increased the escape latency of APP/PS1 mice 24 h after the shock(30 mg/kg TSA,P<0.01;60 mg/kg TSA,P<0.05).Part two Total Salvianolic Acids Regulate Metabolites in the Plasma and Hippocampus of APP/PS1 Transgenic MiceObjective: To observe the changes of the metabolites in the plasma and hippocampus of APP/PS1 mice treated with TSA.Methods:1 Blood samples were collected for the determination of the bloodglucose level via Mut.Q-GDH method using an Accu-Chek Performa glucose meter.The lipids were assessed included CHOL,TG,HDL and LDL.The quantitative determination of the four lipids in the plasma was conducted by the enzymatic color test on Beckman Coulter AU5800 biochemical analyzers.2 The A?42 and A?40 levels in the hippocampus were detected using ELISA.3 Metabolites in the hippocampus of the mice were detected via GC-TOF–MS in four groups.Results:1 A?40 and A?42 increased in the hippocampus of APP/PS1 mice compared with the WT control group(P<0.01).TSA treatment reduced A?40peptide and A?42 peptide(P<0.01)v.s.APP/PS1 mice.The drug efficacy between both doses of TSA had no significant difference(P>0.05).Moreover,the ratio of A?42 to A?40 increased in the APP/PS1 transgenic group(P<0.05),whereas it was unchanged by TSA treatment compared with the APP/PS1group(P>0.05).2 There was no difference in the blood GLUC or plasma TG concentrations among the four groups(P>0.05).High dose of TSA treatment decreased the CHOL level compared with the APP/PS1 group(P<0.05).It is demonstrated that the LDL-C level was increased in the APP/PS1 group compared with the WT group(P<0.05).TSA administration considerably reduced the LDL-C level compared with the APP/PS1 group(P<0.01).The plasma LDL-C level had significant positive correlation with A?42level in the hippocampus of APP/PS1 mice(P<0.01),and the correlation coefficient(r)was 0.875.Similarly,the level of plasma LDL-C was correlated to A?40 level in the hippocampus(P<0.01),and the correlation coefficient of them(r)was 0.728.3 Three hundred fifty-seven peaks were detected in the hippocampus of mice in four groups after GC-TOF-MS detection,with 55 metabolites after the denoising method.The regulation of metabolic pathways in the hippocampus of APP/PS1 mice relevant to TSA predominantly included carbohydrate metabolism,such as sorbitol,glucose-6-phosphate,sucrose-6-phosphate and galactose,vitamin metabolism involved in cholecalciferol and ascorbate in the hippocampus.Part three Total Salvianolic Acids attenuate AD-like pathology in the hippocampus of APP/PS1 MiceObjective:To study how AD-like pathology was regulated in the hippocampus of APP/PS1 mice in three aspects including amyloid plaques,activated microglia and ultrastructure of neurons and capillary endothelial cellin hippocampal CA1 region associated with memory after TSA treatment.Methods:1 HE staining and Nissl's staining were performed to analyze pathology and the number of neurons in the hippocampus of mice in four groups.2 Histochemistry experiment as congo red fluorescence staining and immunohistochemistry method stained with Abeta antibody had been performed to detect the Abeta plaques burdens.3 Ionized calcium-binding adapter molecule 1(Iba1)positive cells in four groups were observed by immunohistochemistry method and the numbers of activated microglia per mm2 area of hippocampus were analyzed.4 The ultrastrutural morphology of neurons and the capillary endothelial cells were observed in hippocampus CA1 region of four groups by TEM.Results:1 TSA treatment reduced the pathological vesicular degeneration in the pathological sections of HE staining in APP/PS1 mice.2 No significant difference was found in the number of neurons per high power field of vision among the four groups in the CA1 region of mice(P>0.05).3 The plaque burdens stained with congo red fluorescence were alleviated in the layer 1,2 and 5 of hippocampal sections(P<0.01)and reduced in the layer 3 and layer 4 in the APP/PS1 mice(P<0.05)after TSA treatment.4 Amyloid ? plaque burdens in the hippocampal sections of four groups stained with Abeta antibody revealed that TSA treatment at doses of 30 mg/kg and 60 mg/kg relieved their corresponding plaque area ratios in APP/PS1 mice(Layer1,2,5,P<0.01;Layer 3: 60 mg/kg TSA,P<0.05;Layer 4: 30 mg/kg TSA,P<0.05;60 mg/kg TSA,P<0.01).5 The number of Iba1 positive microglia per 0.01um2 in hippocampus of APP/PS1 mice increased compared with that of WT control group(P<0.01).The treatment with TSA reversed the impairments in Iba1 positive microglia density(P<0.01)and was comparable with the data of WT control(P>0.05).6 TSA treatments improved the impairment in the ultrastructure ofmitochondrial in the neurons and capillary endothelial cell in the hippocampal CA1 region of APP/PS1 mice.Conclusions: Treatment with TSA induce a remarkable amelioration of learning and memory impairments by protecting the organization of hippocampus,attenuating AD-like pathological changes including amyloid plaques,activated microglia,protecting ultrastructure of neurons and capillary endothelial cells in CA1 region of the hippocampus involved in memory and regulating of metabolites in the hippocampus and plasma.The mechanism of improvement of learning and memory performance by TSA treatment was explored from the aspects of metabolism and pathology.For less relevant reports,this research had a certain originality.