GLP-1 Analogue Ameliorates Cognitive Impairment Induced By MG In Mice

Object Diabetes is a high risk factor for cognitive decline in the elderly.The aim of this study was to investigate the effects of intracerebroventricular injection of methylglyoxal(MG),a metabolite of glucose,on cognitive ability and tau phosphorylation in brain tissue of mice.We investigate the effects of a novel hypoglycemic agent,Liraglutide,which is a GLP-1 analogue,on MG–induced cognitive impairment in mice and to explore potential mechanisms.In vitro,the MG-induced human neuroblastoma cell line(SH-SY5Y)was treated with Liraglutide.It further confirmed the role of Liraglutide in regulating MG-induced tau protein phosphorylation and its mechanism by using inhibitors of related signaling pathways.Methods Part 1:1.To investigate the effect of intracerebroventricular injection of MG on cognitive ability.Eight-week-old male C57BL/6J of SPF grade were randomly divided into Normal saline group,MG low,MG middle and MG high dose group and positive control Aβ group,10 in each subgroup.Normal saline,MG(0.35,0.7,or 1.4 μmol)or Aβ1–42(410 pmol)each in a same volume,were injected by intracerebroventricular injection.Morris water maze was used to explore the effect of different doses of MG on cognitive impairment in mice after 8 weeks.The phosphorylation of tau protein and the expression of the Akt / GSK-3β were detected by Western-blot.2.To investigate the effect of subcutaneous injection of Liraglutide on MG-induced cognitive impairment in mice and its mechanism.Eight-week-old C57BL/6J male mice of SPF grade were randomly divided into vehicle Control group,Liraglutide group,Methylglyoxal group,Methylglyoxal + Liraglutide group,Aβ group and Aβ + Liraglutide group,13-15 in each subgroup.Mice in Methylglyoxal group and Methylglyoxal + Liraglutide group were used intracerebroventricular injection of 0.7 μmol MG to induce cognitive impairment.Mice in Aβ group and Aβ + Liraglutide group were injected with 410 pmol of oligomer Aβ1-42.The learning and memory abilities of mice treated with Liraglutide(25 nmol/kg/day)for 8 weeks were observed by Y-maze and Morris water maze.The ultrastructure of pyramidal cells and synapses in hippocampal CA1 region of mice was observed by transmission electron microscopy.The activities of superoxide dismutase(SOD),glutathione peroxidas(GSH-PX)and content of malondialdehyde(MDA)in the cerebral cortex of mice were measured and used to observe oxidative stress in brain tissue of mice.The expression of p-tau(Ser396)in the cerebral cortex was detected by immunohistochemistry.Westernblot were used to detect the expression of p-tau(Ser202)and p-tau(Ser396)in hippocampus and the activation of Akt / GSK-3β signaling pathway.Part 2:To observe the effect of Liragltuide on hyperphosphorylation of tau protein induced by MG in SH-SY5 Y cells and its mechanism in combination with PI3 K inhibitor Wortmannin of PI3 K / Akt / GSK-3β signaling pathway.After pretreatment with Liraglutide at different concentrations(0,10 nM,100 nM,200 nM)for 24 hours,SH-SY5 Y cell line were induced with different concentrations of MG(0 μM,250 μM,500 μM,750 μM)for 12 h.CCK8 was used to detect the cell viability and apoptosis rate.After that,the appropriate concentration of MG and Liraglutide were used.SH-SY5 Y cell line were randomly divided into Control group,Liraglutide group,Methylglyoxal group,Methylglyoxal+Liraglutide group,Methylglyoxal+Liraglutide+Wortmannin group and Methylglyoxal+GLP-1 group.Westernblot were used to detect the expression of p-tau(Ser202)and p-tau(Ser396),and to verify whether the observed phenomena in animal experiments can be simulated in vitro.Then,we measured the phosphorylation levels of Akt and GSK-3β,which regulate tau signaling pathway.PI3 K inhibitor Wortmannin was used to detect whether tau protein phosphorylation was regulated by Akt / GSK-3β signaling pathway in vitro.Results1.Different doses of MG can damage the cognitive function of mice,but no significant difference among them.Therefore,intracerebroventricular injection medium dose of MG(0.7 μmol)was used as a cognitive impairment model in follow-up experiment.Intracerebroventricular injection of 0.75 μmol MG could promote the phosphorylation of tau by regulting Akt / GSK-3β signaling pathway.2.It suggests that sustained subcutaneous administration of Liraglutide(25 nmol/kg/day)for 8 weeks may protect the cognitive ability in MG-induced mice in behavioral experiment.Furthermore,the results of Transmission Electron Microscopy revealed that Liraglutide was able to reduce the structural degradation of hippocampal CA1 pyramidal cells and synapses in MG-induced cognitive impairment mice.3.Subcutaneous injection of Liragltide has an effect of reducing oxidative stress.Liraglutide can reduce the content of malondialdehyde(MDA)in the cerebral cortex of MG induced cognitive impairment mice and increase the activity of superoxide dismutase(SOD)and glutathione reductase(GSH-PX).Thus Liraglutide could reduce the accumulation of lipofuscin in neuronal cells and mitochondrial structure damage.Furthermore,Liraglutide may reduce the expression of p-tau(Ser202)and p-tau(Ser396)by modulating Akt / GSK-3β signaling pathway in the brain tissue.Similar results were observed in the mice of Aβ+Liraglutide group.4.It showed that viability of SH-SY5 Y cells was increased with pretreated with Liraglutide in vitro experiments.Western-blot analysis showed that Liraglutide decreased the expression of p-tau(Ser202)and p-tau(Ser396)by regulating Akt / GSK-3β signaling pathway.The expression of p-tau(Ser202)and p-tau(Ser396)was increased significantly in SH-SY5 Y cells after administration of PI3 K inhibitor,and the phosphorylation of Akt / GSK-3β was decreased significantly.Conclusion MG may induce tau phosphorylation through Akt / GSK-3β signaling pathway and impair cognitive ability.Liraglutide may decrease the oxidative damage of neuron cells by enhancing the ability of anti-oxidative stress,reduce the expression of phosphorylated tau protein by regulating the activation of Akt / GSK-3β signal pathway,and improve the cognitive ability.Liraglutide may play an important role in Aβ-induced cognitive impairment mice which without disorder of glucose metabolism.Therefore,GLP-1 analogue Liraglutide may has an effect on the improvement of cognitive ablity in mice and the recovery of neurodegenerative diseases.