The Role Of CAMP/CREB In The Improvement Of Aβ25-35-induced Circadian Rhythm Disorder By Liraglutide

Alzheimer’s disease(AD)is a progressive and irreversible neurodegenerative dementia.Disorders of the circadian rhythm can occur early in the disease,which seriously affects human health and life expectancy.Amyloid-beta protein(Aβ)is an important pathological feature of AD.Abnormal accumulation of it causes circadian rhythm disturbance in AD patients and is also one of the main causes of loss of normal rhythmicity of key circadian clock genes.Due to the similar pathological features and clinical manifestations of AD and type 2 diabetes mellitus(T2DM),the use of antidiabetic drugs to treat AD is becoming a hot topic.Liraglutide,a currently available T2 DM therapeutic drug,was selected for this experiment.It has a longer half-life as a natural GLP-1 analogue and retains various physiological roles and advantages of GLP-1.The purpose of this study was to investigate whether Liraglutide can improve circadian rhythm disturbance caused by Aβ25-35 and further explore its related mechanisms.In this study,we performed wheel-running experiments to observe the effect of Liraglutide on the circadian rhythm disturbance caused by Aβ25-35.Liraglutide pretreatment could significantly improve the abnormal expression of Per1 gene/protein rhythm in HT22 cells induced by Aβ25-35.We speculate that Liraglutide can improve circadian rhythm disorder caused by Aβ25-35.The expression of relevant signaling pathway proteins was further examined by ELISA and immunohistochemistry,confirming that Liraglutide could partially up-regulate the decreased of cAMP and p-CREB in hippocampus induced by Aβ25-35.To further prove the protective effect of Liraglutide and its mechanism,the expression of Per1 gene/protein rhythm in HT22 cell was detected by Real-time PCR and Western Blot after adding cAMP inhibitor or CREB inhibitor on the basis of Liraglutide pretreatment,and the results showed that cAMP or CREB inhibitors significantly inhibited the improvement ofPer1 gene/protein expression by Liraglutide,confirming that cAMP/CREB is one of the important molecular mechanisms by which Liraglutide improves Aβ25-35-induced circadian rhythm disturbance..Part I Liraglutide antagonized Aβ25-35-induced disruption of circadian rhythm Objective:To monitor the effect of Liraglutide on the circadian rhythm disturbance induced by Aβ25-35 and the effect of Liraglutide on the abnormal expression of Per1 rhythms gene/protein in HT22 induced by Aβ25-35 at the cellular level.Methods:(1)We injected normal saline,282.9 nmol/kg body weight(bw)Aβ25-35 and 267nmol/kg bw Liraglutide into hippocampus via stereotaxic instrument respectively,and they were randomly divided into Vehicle group,Aβ25-35 group,Liraglutide + Aβ25-35group(Liraglutide pretreatment group)and Liraglutide group.The four groups of mice were then placed in a tread mill set with 12 h : 12 h Light-Dark(LD)for one week followed by a constant darkness(DD)for two weeks,The circadian rhythm of treadmill exercise in four groups of mice was detected by VitalView data collection system and ActiView biorhythm analysis system.(2)At the end of behavioral test,the hippocampus of mice were taken out under darkness conditions.Hippocampus histology slices were prepared to observe the pathological changes.(3)HT22 cells were divided into Vehicle group,Aβ25-35 group,Liraglutide +Aβ25-35 group,Liraglutide group.CCK8 was used to detect whether the decrease of survival rate of HT22 cells induced by Aβ25-35 could be reversed after Liraglutide preconditioning.(4)Real-time quantitative PCR was used to detect the expression of per1 mRNA in HT22 cells of each group at different time points,Western Blot was used to observePER1 protein level at the maximum CT time point of per1 expression,and to observe whether Liraglutide can improve the expression of Per1 gene/protein rhythm in HT22 cells induced by Aβ25-35.Results:(1)After transferred to DD conditions,Aβ25-35 which was injected into hippocampus caused circadian rhythm abnormalities and the circadian rhythm of Liraglutide preconditioned mice returned to normal(P < 0.05).(2)The pyramidal nerves in hippocampal CA1 region of mice in Aβ25-35 group showed obvious pathological changes.Neurons were loosely arranged and contracted and damaged.Liraglutide alleviated the pathological damage of hippocampal CA1 neurons caused by Aβ25-35.(3)Pretreatment with Liraglutide(100 nmol/L)significantly reduced the viability of HT22 cells induced by Aβ25-35(10 μmol/L)(P < 0.05).(4)Liraglutide preconditioning could ameliorate the abnormal expression of Per1 rhythms gene/protein in HT22 cells induced by Aβ25-35(P < 0.05).Conclusion:Intrahippocampal administration of Liraglutide can significantly improve the circadian rhythm disturbance of C57BL/6 mice induced by Aβ25-35 and alleviate the abnormal expression of Per1 gene/protein in HT22 cells.Part Ⅱ Liraglutide Antagonized Aβ25-35-induced Per1 Gene/Protein Rhythm Expression Abnormality in HT22 Cells by cAMP/CREB Objective:To monitor the effect of Liraglutide on the expression of cAMP and p-CREB in the hippocampus and the protective effect of Liraglutide on the expression of Per1gene/protein rhythm in HT22 cell after adding cAMP inhibitor or CREB inhibitor on the basis of Liraglutide pretreatment,and to further investigate the effect of cAMP/CREB in this process.Methods:(1)At the end of behavioral test,the hippocampus of mice were taken out under darkness condition.The cAMP content in the hippocampus was detected by ELISA.Hippocampus histology slices were prepared to detect the expressions of p-CREB.(2)HT22 cells were divided into Vehicle group,Aβ25-35 group,Liraglutide pretreatment group,cAMP inhibitor(SQ22536)+ Liraglutide pretreatment group or CREB inhibitor(KG-501)+ Liraglutide pretreatment group,Liraglutide group,SQ22536 group or KG-501 group.(3)Real-time quantitative PCR was used to detect the expression of per1 mRNA in HT22 cells of each group at different time points,Western Blot was used to observe PER1 protein level at the maximum CT time point of per1 expression,and to observe whether Liraglutide can improve the expression of Per1 gene/protein rhythm in HT22 cells induced by Aβ25-35 through cAMP/CREB.Results:(1)Aβ25-35 induced a decrease in cAMP and p-CREB expression in hippocampal CA1 region of mice,while Liraglutide pretreatment partially upregulated cAMP and p-CREB expression in hippocampus induced by Aβ25-35(P < 0.05).(2)Liraglutide preconditioning could ameliorate the abnormal expression of Per1gene/protein in HT22 cells induced by Aβ25-35,and the improvement was inhibited by the addition of cAMP inhibitor(SQ22526)and CREB inhibitor(KG-501)respectively.Conclusion:Liraglutide pretreatment partially upregulated cAMP and p-CREB expression inhippocampus induced by Aβ25-35.The improvement of Liraglutide was inhibited by the addition of cAMP inhibitor(SQ22526)and CREB inhibitor(KG-501)respectively,and it was very likely that cAMP/CREB is one of the important mechanisms of Liraglutide antagonist Aβ25-35-induced Per1 rhythm gene/protein abnormal expression in HT22 cells.