The Protective Effects And Mechanisms Of GLP-1 Analogue On AD-like Neurodegenration

Objective:To Explore the neuroprotective effects and mechanisms of GLP-1 analogue on AD-like neurodegenerative changes.Methods:1)The human neuroblastoma cell line SH-SY5 Y on logarithmic phase was divided into six groups: normal control(CON)group,treated with PBS for 12h;AD model(W)group,treated with 1?mol/L wortmannin for 12h;drug control groups(D group or P group),10nmol/L Dulaglutide or 50nmol/L GLP-1(7-37)peptide treated for 12h;treatment(D+W or P+W)groups,10nmol/L Dulaglutide and 1?mol/L wortmannin or50nmol/L GLP-1(7-37)peptide and 1?mol/L wortmannin treated for 12h;common intervention(D+W+E or P+W+E)group,10nmol/L Dulaglutide,1?mol/L wortmannin and 10?mol/L Ex9-39 or 50nmol/L GLP-1(7-37)peptide,1?mol/L wortmannin and10?mol/L Ex9-39 treated for 12h;the inhibitor(E)group,treated by 10?mol/L Ex9-39 for 12h.MTT assay was applied to analyse the cell vitality.Western blot assay was used to detect the level of total Tau protein,phosphorylated Tau at different sites and the level of neurofilament heavy and medium chains(NF-H/M).Synaptic protein and phosphorylation of insulin signaling pathway,like PI3 K,GSK-3? and MAPK were analyzed in the same way.Cellular immunofluorescence assay was used to analyze the signal changes of GLP-1.2)20 C57BL/6(Wild Type)mice and 40 triple transgenic APP/PS1/Tau mice were 6 weeks old and divided randomly: wild type mice administrated saline(0.9%w/v)nasally were WT group and treated GLP-1(7-37)peptide nasally were WT+Du group;Tg mice administrated saline(0.9% w/v)nasally were Tg group,administrated GLP-1(7-37)peptide nasally as Tg+Du(N)group,administrated GLP-1(7-37)peptide percutaneously as Tg+Du(P)group and administrated Insulin nasally as Tg+Ins(N)group.Tg+Du(P)and Tg+Ins(N)group had a positive control to Tg+Du(N)group.Mice in different groups mentioned above were received solution twice times everyday until 4 weeks.The dose of treated groups were 10?g GLP-1(7-37)peptide(about 10?l)and 0.4 units Insulin(about 10?l),and that in administrated groups were equivalent saline(0.9% w/v).Morris water maze was applied to analyze the spatial learning and memory ability of mice after 2 weeks,while Body weight and blood sugar were measured everyday.Western blot was used to evaluate the Expression level of phosphorylated Tau and NFs,the level of phosphorylated PI3K-GSK3? and MAPK,the critical enzymes and protein of insulin downstream signaling pathway in the brain tissues.Results:1)Compared with control group,cells in model group and inhibtor group had decreased the cell vitally,increased phosphorylated Tau at serine and the threonine sites(Ser/Thr)262,396,404 and NF-H/M proteins,but in Dulaglutide group and GLP-1(7-37)peptide group had increased the cell vitally,the above indicators were completely opposite.Compared to model group,the cell vitally of drug treatment groups were raised,the indexes mentioned above were descended.But compared with drug treatment groups,both of them had the declined cell vitally and high phosphorylated Expression level by Ex9-39 intervention.The results also show that,compared to control group,there were a decreased level of synaptic proteins and phosphorylated level of PI3K-GSK3? and MAPK in model group and inhibitor group,while an increased level of these indexes in Dulaglutide group and GLP-1(7-37)peptide group.Additionally,compared to model group,drug treatment groups had the growing level of synaptic proteins and phosphorylated level of PI3K-GSK3? and MAPK.After Ex9-39 intervention,these above levels in drug treatment group were obviously degressive.2)Body weight and blood sugar of mice had no significant changes among 6groups.The mean escape latency and path length had much longer than WT Mice,while it had a prominently descending in the number of crossing hidden platform and total time in target quadrant on Tg mice.Moreover,both hyperphosphorylated Tau and NF-H/M proteins were higher in Tg mice than those in WT,while phosphorylated level of PI3K-GSK3? and MAPK were much lower in Tg mice than those in WT.But all these mentioned parameters were improved in Tg mice treated by GLP-1(7-37)peptide and Insulin percutaneously or nasally.Conclusion:1)The GLP-1 analog Dulaglutide and GLP-1(7-37)peptide can improve cell vitality,reduce the hyperphosphorylation of Tau and NFs and increase the synapse expression level by neuroprotective effect on the cellular AD model.2)Intranasal administration of GLP-1(7-37)peptide can improve learning and memory abilities and decrease brain hyperphosphorylation of Tau and NFs by neuroprotective effect on AD mice.