| Objective:To study the neuroprotective effect of icariin?ICA?on APP/PS1/Tau triple transgenic Alzheimer's disease?3×Tg-AD?mice,and explore whether the neuroprotective effect is related to the improvement of cerebral insulin signaling pathway disorders.Methods:3 month old male 3×Tg-AD mice were used as the research object,which were randomly divided into two groups?n=10/group?:3×Tg-AD+Vehicle group and 3×Tg-AD+ICA group;sex-and age-matched wild type?WT?mice were randomly divided into two groups?n=10/group?:WT+Vehicle group and WT+ICA group.After grouping,the mice were orally administered according to the weight of the mice?0.1 mL/10 g?.The 3×Tg-AD+ICA and WT+ICA group were administered ICA 60 mg/kg,and the 3×Tg-AD+Vehicle as well as WT+Vehicle group were administered the corresponding volume vehicle?0.5%Tween-80 in distilled water?,once a day for 5 months.After 5 months,the mice were anesthetized and decapitated,then performed the following experiments:?1?HE staining and Nissl staining were used to evaluate neuronal morphology and the number of survival neurons in cortex of mice.?2?Immunofluorescence was used to observe the neuronal nuclei?NeuN?-positive neurons and?-amyloid?A??aggregation.?3?Western blot was used to detect the expression of NeuN,postsynaptic density protein 95?PSD95?,amyloid precursor protein?APP?,A?1-40,A?1-42,Tau protein phosphorylation at PHF-1?Ser396/404?,Ser199/202,Thr231 and Thr217,and insulin signaling-related proteins including insulin?INS?,insulin receptor?IR?and its phosphorylation?pIR Tyr1361?,insulin receptor substrate-1?IRS1?and its phosphorylation?pIRS1 Ser307 and pIRS1 Ser616?,the regulatory subunit p85 and catalytic subunit p110?of phosphatidylinositol-3-kinase?PI3K?as well as the phosphorylated p85?pPI3K p85 Tyr199/458,pPI3K?,protein kinase B?PKB/AKT?and its phosphorylation?pAKT Ser473?,and downstream glycogen synthase kinase 3?GSK3??and its phosphorylation?pGSK3?Ser9?.Results:?1?Morphological results indicated that cortical neurons in 3×Tg-AD mice showed a significant decrease in number,incomplete structure,and obvious deep staining and nuclear condensation,compared with WT mice,and the pathological phenomena were improved significantly after administration of ICA.?2?The immunofluorescence results showed that in the cortex of 3×Tg-AD mice the number of NeuN-positive neurons was significantly reduced,and A?aggregation was significantly increased,compared with WT mice.After ICA treatment,the number of NeuN-positive neurons were increased,and A?aggregation was reduced.?3?Western blot results indicated that,in the cortex of 3×Tg-AD mice compared with WT mice,the expression of NeuN and PSD95 were abnormally reduced,and APP,A?1-40-40 as well as A?1-42 were significantly increased.After 5 months of ICA treatment,the expression levels of NeuN and PSD95 were significantly increased,and APP,A?1-40-40 as well as A?1-42-42 were significantly decreased.Moreover,the levels of Tau protein phosphorylation at Ser199/202,Thr231 and Thr217 were abnormally increased in cortex of3×Tg-AD mice.After 5 months of ICA administration,the levels of phosphorylation were significantly decreased.However,the expression of PHF-1 was not significantly different among the experimental groups.Additionally,in the cortex of 3×Tg-AD mice,the levels of pIR Tyr1361,pPI3K,AKT,pAKT Ser473 and pGSK3?Ser9 were significantly reduced,and pIRS1 Ser307 as well as pIRS1 Ser616 were significantly increased.After ICA treatment,the levels of pIR Tyr1361,pPI3K,AKT,pAKT Ser473 and pGSK3?Ser9 were significantly increased,and pIRS1 Ser307 and Ser616 were significantly reduced.There were no significant changes in INS,IR,IRS1,p85,p110?and GSK3?among the experimental groups.Conclusion:ICA has neuroprotective effects on 3×Tg-AD mice,and the protective mechanism may be related to its improvement in cerebral insulin signaling dysfunction. |
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