Study On The Effects Of JDYZF On Behavior And Amyloid Deposition And BACE1 Expression In β-cerebral Cortex In Alzheimer’s Disease Mice

Selection of topics:Alzheimer’s disease（AD）disability,high mortality,the pathogenesis is complex,Aβabnormal deposition in the brain is an important pathological change and core link of AD development,previous studies have shown that JDYZF an inhibit plaque deposition and related gene expression AβAD nematode head,A randomized Controlled clinical study showed that the change of MMSE、MoCA、ADL score after 6months of application was significantly better than that of the Control group.Rhizoma Coptidis is the monarch medicine in the detoxification and wisdom prescription,is the main ingredient of the recipe,Play an important role in detoxification.Preliminary liquid chromatography screening showed that JDYZF was rich in polysaccharides,To further explore and find that this substance is the main component of Coptis chinensis,Therefore,this study was designed to explore the protective effect of Coptis chinensis polysaccharide on Aβ25-35 injury PC12,To provide a theoretical basis for the pathogenesis of"poison damage"and the treatment of"detoxification",To provide a research basis for subsequent research.Because previous studies have focused on single-gene,single-celled organisms,Can’t simulate AD disease very well,Hence,APP/PS1 double transgenic mice were selected as the subjects,A mouse that mimics abnormal central nervous system Aβdeposition,Through behavioral and molecular studies of mice,To further evaluate the therapeutic effect of detoxification JDYZF.Objective:The protective effect of CCP on the PC12 of Aβ25-35 injury was studied by cell experiment in vitro.Through behavioral,molecular biology and other research methods,the cognitive function,Aβdeposition,BACE1 expression of related proteins,genes and other aspects of APP/PS1 mice were detected and analyzed,and the mechanism of JDYZF on APP/PS1 mice was discussed,which provided reliable research evidence for the popularization and application of traditional Chinese medicine treatment AD.Methods:1.This study explored the mechanism of JDYZF from in vitro and animal experiments.Mitochondrial activity,cell viability and apoptosis of Aβ25-35 induced PC12 cell injury model were determined by MTT reduction,Hoechst33258 fluorescence staining,To explore the protective effect of CCP on PC12 cells.Animal experiment part selected APP/PS1double transgenic mice,Randomly divided into five groups,They were model group（APP/PS1 group）,Donepezil hydrochloride group（Donepezil group）,low dose group（JDYZFL group）,medium dose group（JDYZFM group）and high dose group（JDYZFH group）,16 mice per group,Sixteen C57 mice were selected as normal control group（Control group）,Gavage at 8:30 am,The daily gavage drugs and doses were,Normal group and Model group 0.5%CMC Solution 0.20 g·kg-1·d-1;Control group:0.30 g·kg-1·d-1;Donepezil Hydrochloride Drug concentrations in High,Medium and Low dose groups were0.60 g·kg-1·d-1,respectively 0.3 g·kg-1·d-1,0.15 g·kg-1·d-1.End of treatment for 6months.2.Aβ25-35 damaged PC12 cells,the neuroprotective effects of CCP were evaluated by measuring cell viability,LDH release rate,apoptosis rate,and the level of mitochondrial membrane potential.3.Through the water maze,Document the effect of JDYZF on learning and memory ability of APP/PS1 mice.4.ELISE and Immunofluorescence were used to detect the changes of Aβlevels in mice,To observe the effect of JDYZF on Aβdeposition in APP/PS1 mice.5.Use PCR methods to detect changes in brain tissue SIRT1、NF-κB、BACE1 gene levels,Western blot method was used to detect the changes of SIRT1、NF-κB、BACE1protein in Aβof brain tissue,To observe the effect of JDYZF on the SIRT1、NF-κB、BACE1 of AMPK/SIRT-PPARγ-PGC1α-BACE1 transporter pathway in APP/PS1 mice.Results:1.The effects of CCP on Aβ25-35-induced PC12 cell injury were observed in vitro PC12 cell viability assay showed that PC12 cells were treated with 5-50μM Aβ25–35for 24 hours,cell viability decreased from 24%to 61%with increasing Aβ25–35concentration.When the Aβ25–35 was in the concentration range of 5 to 50μM,the LDH release in the cells increased to about 130-160%of the Control group.Treatment of PC12cells with different concentrations of CCP（5 to 200μg/ml）for 24 hours,no significant cell loss was observed.The results of Hoechst33258 staining and FCM determination showed that the nucleus had significant concentrated nucleus and apoptotic cell formation under fluorescence microscope.As intact as observed in untreated PC12 cells,Round and relatively large nuclei,50μMAβ25–35 exposure The cells at 24 hours showed typical apoptotic features,Including chromatin condensation,nuclear fragmentation and the emergence of apoptotic bodies.CCP different concentrations（5,25,50,100,200μg/ml）can effectively reverse apoptosis after treatment,Among them 100μg/ml effect is most obvious,FCM analysis showed exposure to50μMAβ25–35 alone 24 hours leading to 58.88±6.12%apoptosis,this was significantly different from the values of 7.21±0.82%in the normal Control group（P<0.01）.With100μg/ml of CCP,Apoptosis decreased to 12.51±1.32%.The Aβinduced mitochondrial dysfunction was detected by JC-1 the ratio of red fluorescence to JC-1 green fluorescence.The results showed that the cells were exposed to 50μM,After 24 hours of Aβ25–35,Compared with the normal Control group,A significant reduction in JC-1 red fluorescence ratio,The JC-1 green fluorescence ratio was increased（P<0.01）.PC12 cells pretreated with CCP（100μg/ml）Aβ25–35 significantly offset the membrane potential loss caused by Aβ25–35 injury,JC-1 changed from 22.1%of Aβ25–35 treated PC12 cells to 84.3%green fluorescence,close to the normal Control group（P>0.05）.The results showed that the expression of cytochrome C in cytoplasm increased after 24 hours PC12 50μMAβ25–35treatment,whereas PC12 cells pretreated with CCP（100μg/ml）significantly attenuated cytoplasmic cytochrome C.released by mitochondria2.To observe the effect of JDYZF on learning and memory ability of APP/PS1transgenic miceThe results of the water maze experiment showed that the incubation period,path length and swimming distance of the mice in each group were shortened with the backward shift of the experimental days,indicating that the localization memory ability of the limited platform of the mice in each group was improved with the increase of the training times.Compared with the normal group,The escape latency,path length and swimming distance of the model group were significantly prolonged,A significant difference（P<0.01）,The first time to reach the platform was significantly longer,The number of stopovers is significantly reduced,difference was significant（P<0.01）.Compared to the model group,The escape latency,path length,swimming distance of Donepezil hydrochloride group,JDYZFL group,JDYZFM group were shortened,A statistically significant difference P<0.05,P<0.01),The first time you reach the platform,Increased number of target stays,A statistically significant difference P<0.05,P<0.01).Compared with Donepezil hydrochloride,JDYZFL group had the same effect,JDYZFM group was slightly superior to Donepezil hydrochloride group,difference was statistically significant（P<0.05）.The results of localization navigation and spatial search of 12-month-old mice treated with 6-month intervention were better than those of 9-month-old mice treated with 3-month intervention,difference was statistically significant（P<0.05）.3.To observe the effect of JDYZF on Aβdeposition in the brain of APP/PS1 double transgenic miceCompared with normal Control group,the results as follows:soluble and insoluble Aβin Aβof model group Aβ_1-40and Aβ_1-42The positive number of Shen increased significantly（P<0.01）.Compared with the model group,the soluble and insoluble Aβin Aβof JDYZFL group,JDYZFM group and Donepezil hydrochloride group were significantly increased Aβ_1-40and Aβ_1-42The amount of deposition decreased in different degrees（P<0.05,P<0.01）.Compared with Donepezil hydrochloride group,soluble and insoluble Aβin Aβof JDYZFM group Aβ_1-40and Aβ_1-42and the amount of deposition decreased,the difference was statistically significant（P<0.05）.The expression of Aβin Aβof mice by Immunofluorescence assay showed that only trace Aβ,were produced in normal group。The expression of Aβin the model group was significantly increased,and the soluble and insoluble Aβof 12 month old intervention group mice Aβ_1-40and Aβ_1-42.The positive number of sinking was significantly lower than that of 9months old mice.The Immunofluorescence labeling showed that the Aβdeposition in the intervention group was significantly reduced and the Aβdeposition in the model group was significantly increased.4.Regulation of BACE1 Expression（1）PCR test resultsPCR test results of each group showed that compared with normal Controls,Expression of BACE1/GAPDH、NF-κB/GAPDH in Aβof model mice was significantly increased,SIRT1/GAPDH significantly reduced,（P<0.01）,Compared to the model group,The levels of BACE1/GAPDH、NF-κB/GAPDH expression in Aβof mice of Donepezil Hydrochloride Group,JDYZFL Group and JDYZFM Group decreased in varying degrees,SIRT1/GAPDH expression levels increased,P<0.05,P<0.01),JDYZFH group had statistical difference in NF-κB/GAPDH expression in the morning.Compared with Donepezil hydrochloride,The BACE1/GAPDH、NF-κB/GAPDH expression content of JDYZFL group and JDYZFM group decreased in different degrees,difference was statistically significant（P<0.05）.PCR results confirmed that other drug intervention groups except JDYZFH group could play a neuroprotective role by increasing the content of SIRT1mRNA and reducing the expression of NF-κBmRNA、BACE1mRNA,thus inhibiting the production of Aβ.expression of hippocampal body Aβdecreased in each treatment group.The expression of SIRT1mRNA in each group was increased and the expression of NF-κBmRNA、BACE1mRNA decreased in all groups except the model group and the normal Control group compared with those in the continuous gavage group for 3 months（P<0.05）（2）Westorn blot test resultsWestern blot test results of each group showed that compared with normal group,Expression of protein SIRT1 in Aβof model mice decreased significantly,NF-κB、BACE1protein expression was significantly increased（P<0.01）.Compared to the model,The expression of Donepezil,JDYZFL and JDYZFM groups decreased,There was significant difference in SIRT1 protein expression（P<0.05）.Compared with donepezil hydrochloride,The expression of NF-κB protein in JDYZFL group,JDYZFM group,difference was statistically significant（P<0.05）,There was no significant difference in SIRT1、BACE1protein expression in JDYZFL group,SIRT1 expression in JDYZFM group improved,BACE1 protein expression decreased slightly,difference was statistically significant（P<0.05）.After 3 months and 6 months of intervention,the expression of AβAβdecreased in each treatment group.The expression of protein in each group for 6 months was increased and the expression of NF-κB、BACE1 protein was decreased in all groups except the model group and the normal Control group,compared with the mice SIRT1 3 months continuous gavage（P<0.05）.Conclusion:1.The mechanism of CCP protection of Aβ25-35 damaged PC12 cells may be related to enhancing cell viability,inhibiting apoptosis,reducing LDH release,blocking membrane potential loss,preventing mitochondrial cytochrome c release and repairing mitochondrial dysfunction.2.JDYZF results of positioning navigation experiment and spatial search experiment of APP/PS1 double transgenic mice could be improved in different dose groups,indicating that JDYZF can improve the spatial learning and memory ability of mice.3.JDYZF can reduce the abnormal deposition of Aβin the cerebral cortex of APP/PS1double transgenic mice to varying degrees.4.JDYZF inhibit abnormal expression of BACE1,its mechanism may be achieved by regulating the expression of related factors in the AMPK/SIRT1-PPARγ-PGC1α-BACE1signaling pathway in the brain.