Phenethylalcohol Glycosides Of Cistanche On Neuroprotective Effects Of APP/PS1 Double-transgenic AD Mouse Model And Its Mechanism

Objectives:1.Study the effects of phenylethanoid glycosides of cistanche on the behavior and the ability of learning and memory in AD model mouse which transfected with APP/PS1 double transgenic.2.Investigate the effect of phenylethanoid glycosides of cistanche on the degree of Aβon AD model mouse.3.Discuss the mechanism of Insulin signal transduction pathway in phenylethanoid glycosides of cistanche antagonistic AD.4.Optimized of 2 AD cell models in vitro based on the Aβhypothesis and oxidative stress hypothesis,validated the protective effect of phenylethanoid glycosides of cistanche.Method:1.APP/PS1 double transgenic mices and wild-type C57 mices were used as experimental platform in vivo.The transgenic mices were randomly divided into normal group,model group,donepezil group（dosage:0.65 mg/kg）,phenylethanoid glycosides of cistanche divided into high,medium and low dose group（dose was 250、125、62.5 mg/kg）,cistanches acteoside dose group（125 mg/kg）,each group of 10 mices,the mice were treated with intragastric administration at the age of 6^th month,continuous administration for 3 months.The model group and normal group were given equal volume of distilled water.The ability of learning and memory in mice was tested by facial tissue nesting method,Morris water maze method and step down test,to evaluate the effect of phenylethanoid glycosides of cistanche in cognitive function of AD model mouse.2.HE staining was used to observe the changes of brain hippocampus,Immunohistochemistry and Western-blot method was used to test the protein expression of Aβ_1-42-42 and Aβ_1-40.Inferred the effect of phenylethanoid glycosides of cistanche on the degree of Aβin AD model mouse which transfected with APP/PS1 double transgenic.3.Immunohistochemical method and Western-blot method were used to investigate the effect of phenylethanoid glycosides of cistanche on the distribution and the protein expression levels of InR、IRS-1、IGF-1R、PI3K、p-PI3K、AKT、p-AKT and other related molecular in insulin signaling pathway,to evaluate the regulation of insulin signaling pathway.4.Using PC12 cell as the model carrier,select the most neurotoxic Aβ_1-42-42 fragment and the most representative H₂O₂ as induction factors,optimization of 2 experimental models of AD in vitro,Cell viability was measured by MTT assay,enzyme assay determination of LDH leakage rate,determination of MDA content by TBA,to investigate the protective effect of phenylethanoid glycosides of cistanche on damage model.Results:1.Nesting experiment show:compared with the model group,phenylethanoid glycosides of cistanche groups with the dose increased,nesting score improved significantly.Morris water maze show:compared with the model group,phenylethanoid glycosides of cistanche treatment group mice escape latency decreased significantly（p<0.05）,the residence time in the target quadrant was significantly increased（p<0.05）.the swimming trajectories of mice were linear or directional,the number of mice crossing the platform was significantly increased（p<0.05）,the step-down test results showed that compared with the model group,phenylethanoid glycosides of cistanche treatment group mice significantly reduced the number of errors in platform（p<0.05）.2.Phenylethanoid glycosides of cistanche can improve the hippocampus of APP/PS1 double transgenic mice damage,Immunohistochemical results showed that:Compared with the model group,the number of a Aβ_1-42-42 and Aβ_1-40 positive cells in hippocampus CA1 area of donepezil group,phenylethanoid glycosides of cistanche in each dose group and acteoside intervention group were significantly decreased（p<0.05）,The results of Western-blot showed that the expression of each protein was consistent with immunohistochemistry.3.Immunohistochemical results showed that:Compared with the model group,the number of InR,IRS-1,IGF-1R positive cells in hippocampus CA1 area of donepezil group,phenylethanoid glycosides in each dose group and acteoside intervention group were significantly decreased（p<0.05）.Western-blot results showed that compared with the model group,the expression of InR,IRS-1,IGF-1R protein in hippocampus CA1 area of donepezil group,phenylethanoid glycosides in each dose group and acteoside intervention group was significantly decreased（p<0.05）,PI3K,p-PI3k,AKT,p-AKT protein expression was significantly up-regulated（p<0.05）.4.The best modeling concentration of the Aβ_1-42-42 damage model is 0.5μM,damage for 48 h.The best conditions of H₂O₂damage model is injury with PBS as solvent,25μM concentration damage for 24 h.Compared with the model group,phenylethanoid glycosides of cistanche and acteoside intervention group,echinacoside intervention group can make the cell survival rate was significantly increased（p<0.05）,LDH leakage rate decreased（p<0.05）,MDA content decreased（p<0.05）.Conclusions:1.Pathological and behavioral characteristics of APP/PS1 double transgenic mice,is an ideal AD platform for research in vivo.The phenylethanoid glycosides of cistanche can significantly improve the cognitive dysfunction of the mouse model.2.The phenylethanoid glycosides of cistanche by reducing the production of Aβ_1-42 and Aβ_1-40 in the hippocampus of the model mice,thus affecting the Aβcascade to protect neurons,further play the role of antagonistic AD.3.The phenylethanoid glycosides of cistanche regulated the upstream and downstream key targets,improved the impaired insulin signaling pathway,regulated brain energy metabolism,and ultimately play a neuroprotective role.This study was based on the cellular and molecular level,to investigate the The phenylethanoid glycosides of cistanche of anti AD specific mechanism.Lay the foundation for the development of phenylethanoid glycosides of cistanche as a new drug,which anti-AD based on mother nucleus structure of Phenethylalcohol glycoside.4.2 kinds of AD in vitro experimental models have advantages and disadvantages,phenylethanoid glycosides of cistanche showed the obvious injury protection,confirms the two classic AD theory of theβ-amyloid protein and oxidative stress,at the same time showed potential anti AD activity.