The Protective Effects Of DPP-4 Inhibitors On AD-like Neurodegeneration And It's Mechanism

Objective:To explore the protective effects of dipeptidyl peptidase-4(DPP-4)inhibitor on AD-like neurodegenerative changes and its mechanism.Methods:1)Wild type C57BL/6 mice and triple transgenic APP/PS1/Tau mice were divided into four groups randomly,named WT group,Tg group,Tg+SAX group and Tg+SIT group and each group contained 15 animals.They began to receive sitagliptin and saxagliptin at 10 month of age after adapting the environment.The dose of saxagliptin(2.6 mg/kg)and sitagliptin(3.5 mg/kg)(about 260?l)or equivalent saline(0.9% w/v)were administered daily for 8 weeks by gastric gavage.Body weight and fasted plasma glucose were measured.Morris water maze was applied to evaluate the spatial learning and memory ability of mice.The deposit of A?42 was detected by immunohistochemistry staining.NFT and neurodegenerative neurons were labeled by modified Bielschowsky's silver staining,Fluoro-Jade B and Nissl staining,respectively.The microtubule binding assay was applied to analyze assembly function of Tau binding to microtubule.And western blot and immunofluorescence staining were used to measure the expression level of GLP-1 and its receptor GLP-1R,the level of phosphorylated and O-Glycosylated Tau and neurofilament heavy and medium chains(NF-H/M),synaptic proteins,phosphorylation of insulin receptor substrates-1 and the level of phosphorylated PI3 K,Ak,GSK-3?,CREB and JNK,ERK,the critical enzymes and protein of GLP-1 downstream signaling pathway in the brain tissues.2)The human neuroblastoma cell line SH-SY5 Y on logarithmic phase was divided into six groups: wortmannin intervention group(W group,treated with 0.03?mol/L wortmannin for 12 hrs),DPP-4I intervention group(DPP-4I group,treated with 10 ?mol/L DPP-4I for 12 hrs),both DPP-4I and wortmannin intervention group(DPP-4I+W group,pre-treated with 10 ?mol/L DPP-4I for 2 hrs,then 0.03 ?mol/L wortmannin for 12 hrs),DPP-4I,wortmannin and Ex9-39 intervention group(DPP-4I+W+Ex9-39 group,pre-treated with 10 ?mol/L Ex9-39 for 2 hrs,then 10?mol/L DPP-4I for 2 hrs followed by 0.03 ?mol/L wortmannin for 12 hrs),Ex9-39 intervention group(Ex9-39 group,treated with 10 ?mol/L Ex9-39 for 12hrs)and control group(CON group,treated with PBS contained 1‰ DMSO for 12 hrs).MTT assay was used to detect the cell vitality.Western blot assay was applied to analyze the level of total Tau protein and phosphorylated Tau at different sites,the level of phosphorylated NFs and phosphorylation of PI3 K,Akt,GSK-3?,the critical enzyme of GLP-1 downstream signaling pathway.Results:1)Body weight and fasted plasma glucose of mice were comparable with no significant differences among four groups throughout the study period.Compared with WT mice,the Tg mice had longer mean escape latency and path length while the number of crossing hidden platform and time spent in target quadrant were significantly decreased.Additionally,more deposition of A?42,neurodegenerative neurons and NFT were observed in brain tissues of Tg mice than WT mice.The results also showed that both hyperphosphorylated Tau and NFs proteins had deceased O-Glycosylated protein and the Tau bound to microtubules was lower in Tg mice compared to WT mice.Furthermore,there were a reduction of the levels of synaptic proteins and phosphorylated IRS-1,PI3 K,Akt,GSK-3?,CREB and ERK and a elevation of the levels of phosphorylated JNK discovered in Tg mice in comparison with WT mice.However,comparing with Tg mice,the all above mentioned parameters were improved in sitagliptin-treated and saxagliptin-treated Tg mice.2)Comparing to the control cells,cells treated with wortmannin had the decreased cell vitality and increased phosphorylated Tau at serine and threonine sites199/202,231 and 396 and NFs protein.DPP-4 inhibitor improved the cell vitality and the phosphorylated Tau and NFs,however this improvement can be canceled by Ex9-39 treatment.The expression levels of phosphorylated PI3 K,Akt and GSK-3?increased significantly in DPP-4 inhibitor-treated cells compared with control cells,while wortmannin-treated cells decreased.Additionally,the expression levels of phosphorylated PI3 K,Akt and GSK-3? were significantly increased in DPP-4 inhibitor and wortmannin co-treated cells than in DPP-4 inhibitor and wortmannin co-treated cells pre-treated by Ex9-39.Conclusion:DPP-4 inhibitors can significantly improved the impaired spatial learning and memory ability and AD-like neurodegeneration,which is possibly relative to the improvement of GLP-1 signaling pathway.