| ObjectiveThis study was aim to observe the.effects of different doses of Danggui Shaoyao San(DSS)on the learning and memory ability of D-galactose model mice.Then the effective concentration of DSS were applied to explore the effects of Danggui shaoyao san detection and its’ blood drug(DSC)and water drug(FBZ)on learning and memory ablitity in APP/PS1 mice model.After treatment with DSS,DSC and FBZ,furher mechanisms were explored,then it may can apply a reference methods for AD research.MethodsFirst,in vitro experiments(A)D galactose model experiment1.Behavioral test:70 male Kunming mice weighing 35g were randomly divided into normal group,model group,Danggui Shaoyao San low dose group,Danggui Shaoyao San high dose group and positive control group.The mice in the model group were treated with subcutaneous injection of D-galactose at a concentration of 100 mg/kg.The mice in the normal group and the treatment group were treated with subcutaneous saline in the neck.After 4 weeks of modeling,the treatment group was given 1.6 g/kg/d and 6.4 g/kg/d of Danggui Shaoyao San for intragastric treatment,and the positive control group was given 3 mg/kg/d edaravone for intraperitoneal injection.The treatment period was 8 weeks,and the model and treatment group was still given D—galactose in the course of treatment.At the end of the administration,Morris water maze,Y maze and conditional fear experiments were used to detect the cognitive function of the animals.(B)APP/PS1 mice model experimentI.Behavioral experiment:70 mice of 6 months old APP/PS1 double transgenic mice were randomly divided into model group,Danggui Shaoyao Powder(DSS)group,nourishing blood circulation(DSC)and spleen dampness(FBZ)group,14 mice with the same age as transgenic negative mice were used as negative control group.The dose of DSS group is 6.4 g/kg/d;Corresponding to the total dose,the dose of DSC group is 4.02 g/kg/d;Corresponding to the total dose,the dose of FBZ group is 2.38 g/kg/d,positive control group was given 3 mg/kg/d Donepezil for intragastric administration,negative control group and model group were treated.with ultrapure water,all the treatment is once a day,the administration period was 8 weeks.At the end of the administration,Morris water maze,Y maze and fear experiments were used to detect the cognitive function of the animals.2.The expression of Aβ1-42 in the cerebral cortex and hippocampus of mice were detected by immunohistochemistry,the level of Aβ1-42 in the cerebral cortex was detected by ELISA.Immunohistochemical method was used to observe the expression of amyloid plaques in the brain of mice and the state of astrocytes.The levels of SYP,BDNF,NGF,APP,PSl,BACE1,NEP,IDE,LRP1,RAGE in the brain of mice were observed by Western blotting.The neuron survival conditong in the brain was observed by Nissl’ s staining.Reults:(A)D galactose model experiment1.Effect of Danggui Shaoyao San on learning and memory ability of D galactose mice(1)Morris water maze results suggest that the escape latency of model mice was significantly longer than that of normal group(p<0.05)after 12 weeks of D galactose administration,while low dose,high dose of Danggui Shaoyao San and edaravone treatment group could significantly increase the escape latency of model mice(p<0.05).Morris water maze space exploration results suggested that after given 12 weeks of D galactose model,the first time of crossing the target of model mice were significantly longer than normal group(P<0.01),the time stay in the target quadrant of model group were much lower than normal group(P<0.01).The time of the first corssing the target of model group was significantly higher than that of the normal group(P<0.05).The high dose of DSS and the positive control agent edaravone could significantly improve the above indexes(P<0.05 or P<0.01).(2)Y maze results suggested that after 12 weeks of D galactose administration,the alternation ratio in the three arms of model group were significantly shorter than the control group(P<0.05),while high dose of DSS and edaravone treatment group could increased the alternation ratio of model group(P<0.05).(3)The results of Conditional fear experiment showed that after 12 weeks of D galactose administration,the freezing time of model group were lower than control group(P<0.05),the high dose of DSS group and edaravone treatment group(P<0.05)could increase the freezing time of model group(P<0.05).(4)We found that 6.4 g/kg/d dose of Danggui Shaoyao San to improve the learning and memory ability of D-galactose mouse model is more obvious.(B)APP/PSl model experiment1.Effect of Danggui Shaoyao San on the ability of learning and memory of APP/PS1 mice(1)Morris water maze results suggest that the escape latency of APP/PSl model group was significantly longer than that of normal control group(P<0.05),DSS and FBZ treatment group could increase the escape latency of APP/PS1 mice model(P<0.05).(2)Compared with the normal control group,the alternation ratio of APP/PS1 model group were significantly decreased in the three arms(P<0.01),while DSS,DSC,FBZ and positive drug Donepezil group could increase the percentage of the alternation ratio of model group(P<0.05 or P<0.01).(3)Compared with the normal control group,the activity time of APP/PSl model group was significantly shorter in the central area(P<0.01),whilel DSS,DSC,FBZ group and positive drug Donepezil group could increase the time of model mice in the central region(P<0.05 or P<0.01).2.Effects of Danggui Shaoyao San on Biochemical Indexes of APP/PS1 Model Mice(1)The results of thioflavan S staining showed that no significant amyloid plaques were found in the hippocampus and cortex of the normal control group.Compared with the control group,the mice in the 8—month-old APP/PS1 model group had higher effects on the cortex and hippocampus(P<0.01).The deposition of amyloid plaques in the model mice was significantly reduced by DSS and DSC,FBZ decoction group and positive drug donepezil group(P<0.01).(2)Immunofluorescence results showed that Aβ1-42 positive staining was not found in the brain of the same month and same background non-transgenic mice.The expression of Aβ1-42 in the cortex and hippocampus of mice in the APP/PS1 model group was significantly increased(P<0.01)and the astrocytes in model mouse brain are in an activated state.While DSS and DSC,FBZ decoction group and positive drug donepezil group could downregulate the level of Aβ1-42 and inhibit the activation of astrocytes.(3)Elisa results suggest that the secretion of Aβ1-42 in the mice of APP/PS1 model group was significantly higher than that of the control group(P<0.01).The secretion of Aβ1-42 in the brain of model mice was significantly down-regulated by FBZ,DSC decoction group and positive drug donepezil group(P<0.05).(4)Compared with the control group,the number of positive cells in the cortex and hippocampal CA1,CA3 and DG(P<0.01)in the model group was significantly lower than that in the control group.The number of nissle positive cells were increase by treatment with DSS and DSC,FBZ decoction group and positive drug donepezil group(P<0.01).(5)Compared with the control group,the expression of APP,PS1 and BACE1 protein in the model group was significantly higher than that in the control group(P<0.01).The expression of NEP and IDE in the model group was significantly lower than that in the control group(P<0.05).Compared with the model group,the expression of APP,PS1,NEP and IDE did not changed in the treatment group(P>0.05).The expression of LRP1 protein in the model group was significantly lower than that in the control group(P<0.01).The expression of RAGE was significantly decreased in DSS group and DSC,FBZ group and Donepezil group(P<0.01).Compared with the control group,the expression of SYP,BDNF and NGF in the model group was significantly lower than that in the control group(P<0.01),and the expression of DSS(P<0.01).The expression of SYP,BDNF and NGF was significantly increased in the treatment group(P<0.01).(6)We found that both DSS and DSC and FBZ groups significantly reduced the deposition of amyloid plaques in the brain of APP/PS1 mice,down-regulated the level of Aβ1-42 in the brain of mice,and improved the neuron survival condition of APP/PS1 model mice brain.The levels of SYP,BDNF and NGF in the brain of model mice were improved by DSS,DSC and FBZ.What’ s more,DSS and DSC and FBZ could inhibite the activation of astrocytes in the brain of APP/PS1 mice.However,the level of APP,PS1 and BACE in the APP pathway of APP/PS1 model mice was not significantly regulated by DSS,DSC and FBZ decoction groups.There was no obvious regulation on the expression of A β-degrading protein NEP and IDE by DSS,DSC and FBZ decoction groups.DSS,DSC and FBZ decoction groups could up-regulate the level of LRP1 which is responsible for transporting Aβ into brain,down-regulate the level of RAGE which is responsible for transporting Aβ out of brain in APP/PS1 mouse model.ConclusionDSS,DSC and FBZ could ameliate the cognitive deficit of APP/PS1 mice.The mechanism may be association with its effction of up—regulating Aβ transporting the level of brain protein LRP1,down-regulating Aβinto brain protein RAGE Level,thereby reducing the deposition of amyloid plaques in the brain,inhibiting the excessive activation of asytes in the brain on the neuron cell damage. |
PDF Full Text Request