D-Ser2-Oxyntomodulin Ameliorates Aβ31-35 Induced Circadian Rhythm Disorder And The Mechanism

The maintenance of normal function of the circadian system is indispensable in all areas of life.Circadian rhythm disorder(CRD),which occurs in the early stage of Alzheimer’s disease(AD),has a serious impact on the quality of patients’ life and aggravates the development of AD.Therefore,it is of great significance to explore the cure against circadian rhythm disorder in AD.Amyloid β-protein(Aβ)is believed to be closely related to formation of circadian rhythm disorder in AD patients.Many similarities of pathogenesis and pathological manifestations between type 2 diabetes mellitus(T2DM)and AD have been identified,which provides a new direction for the treatment of AD.The glucagon-like peptide-1/glucagon dual receptor agonist Oxyntomodulin(OXM)is a new kind of drug for T2 DM.D-Ser2-Oxyntomodulin(Oxy),a new OXM analogues whose neuroprotective effect has been already confirmed in recent years and GLP-1 receptor may exerts improtant effect in it.However,whether Oxy can improve circadian rhythm disorders in AD is still unknown.In this study,we confirmed the improvement effect of Oxy on circadian rhythm disorder and abnormal rhythmic expression of circadian clock genes Bmal1 and Per2 induced by Aβ31-35 in vivo by using wheel running behavior experiments and Real-time PCR.In vitro,Oxy antagonized the toxicity of Aβ31-35 on the hippocampal HT22 neuronal cells in a dose-dependent way.Real-time PCR and Western Blot experiments were applyed to show that Oxy significantly improved abnormal rhythmic expression of circadian clock genes Bmal1 and Per2 induced by Aβ31-35.Using lentivirus to interfere the GLP-1 receptor gene in HT22 cells,it was found that GLP-1 receptor gene silence induced aberrant expression of Bmal1 and Per2,and the neuroprotective effect of Oxy against Aβ was abolished.In summary,Oxy ameliorates Aβ31-35 induced circadian rhythm disorder and abnormal rhythmic expression of circadian clock genes and GLP-1receptor plays an important role in it,which may provide a new target for treatment of circadian rhythm disorders in AD patients.Part Ⅰ Aβ31-35 induces circadian rhythm disorder and abnormal expression ofcircadian clock genes Objective:To verify that Aβ31-35 can induce circadian rhythm disturbance in mice,abnormal expression of circadian clock genes in mouse hippocampus and abnormal expression of circadian clock gene in HT22 cells of mouse hippocampus.Methods:The 6-8 weeks old male C57BL/6 mice were randomly divided into 2 groups: control group and Aβ31-35 group.We used the wheel running behavior experiment to observe the circadian running rhythms of mice,and to detect the changes of locomotor activity,free-running period and subject day/night activity in mice.Using Real-time PCR to detect the rhythmic changes of circadian clock genes Bmal1 and Per2 in hippocampus.The mouse hippocampal HT22 neuronal cells were divided into control group and Aβ31-35 group.CCK-8 cytotoxicity test was used to detect the cell survival rate under 2.5,5 and 10μM Aβ31-35.Real-time PCR and Western Blot were applyed to detect the changes of circadian clock gene expressions at different time points under 5 μM Aβ31-35 in HT22 cells.Results:(1)Compared with control group,the running activities of mice in Aβ31-35 group is relatively scattered and the pattern of sleep/activity becomes irregular.Besides,we observed decreased locomotor activity,prolonged free-running period and increased activity amount during subject day of mice induced by Aβ31-35.(2)Compared withcontrol group,phase delay of circadian clock genes Bmal1 and Per2 mRNA expression in Aβ31-35 mice hippocampus was observed,and the expression significantly decreased in CT20 and CT16 respectively.(3)2.5,5 and 10 μM Aβ31-35 decreased survival rate of HT22 cells significantly compared with control group.(4)Compared with control group,we observed abnormal expression of Bmal1 and Per2 mRNA and protein in HT22 cells after Aβ31-35 administration,which decreased obviously in CT20 and CT16 respectively.Conclusion:Aβ31-35 induces circadian rhythmic disorder and abnormal expression of circadian clock genes Bmal1 and Per2 in mice.Part Ⅱ D-Ser2-Oxyntomodulin(Oxy)improves circadian rhythm disorder and abnormal expression of circadian clock genesObjective:To verify the effect of D-Ser2-Oxyntomodulin(Oxy)on the circadian rhythm disorder,the abnormal expression of circadian clock genes in mice hippocampus and the abnormal expression of circadian clock genes in HT22 cells induced by Aβ31-35.Methods:The 6-8 weeks old male C57BL/6 mice were randomly divided into 4 groups: control group,Aβ31-35 group,Oxy pretreatment group(Oxy+Aβ31-35 group)and Oxy group.We used the wheel running behavior experiment to observe the circadian running rhythms of mice,and to detect the changes of locomotor activity,free-running period and subject day/night activity in mice.Using Real-time PCR to detect the rhythmic changes of circadian clock genes Bmal1 and Per2 in hippocampus.The mouse hippocampal HT22 neuronal cells were divided into four groups as mentioned above.CCK-8 cytotoxicity test was used to detect the influence of Oxy on cell survival rate.Real-time PCR and WesternBlot was applyed to detect the influence of Oxy on abnormal expressions of Bmal1 and Per2.Results:(1)Compared with Aβ31-35 group,the mice in Oxy pretreatment group performed more regular running activities,more fixed pattern of sleep/activity,higher locomotor activity,shorter free-running period and decreased activity amount during subject daily.(2)Compared with Aβ31-35 group,the expression of Bmal1 and Per2 mRNA in the mouse hippocampus of Oxy pretreatment group recovered to a certain extent,with significantly increased expression at CT20 and CT16 respectively.(3)1,10,100 and 200 nM Oxy pretreatment improved the survival rate of HT22 cells after Aβ31-35 administration,and100 and 200 nM doses significantly increased the cell survival rate.(4)Compared with Aβ31-35 group,the abnormal expression of Bmal1 and Per2 mRNA and protein in HT22 cells of Oxy pretreatment group was ameliorated,with the expression significantly increased in CT20 and CT16 respectively.Conclusion:Oxy improves the circadian rhythm disorder and the abnormal expression of circadian clock genes Bmal1 and Per2 induced by Aβ31-35 in both vivo and vitro.Part Ⅲ GLP-1 receptor invovles in Oxy improving Aβ31-35 induced abnormal expression of circadian clock genes in HT22 cellsObjective:To observe the effect of GLP-1 receptor(GLP-1R)gene silencing on the circadian clock genes expression in HT22 cells,and observe whether Oxy can improve the abnormal expression of circadian clock genes induced by Aβ31-35 when GLP-1R gene have been interferenced.Methods:Lentivirus vectors containing GLP-1R-shRNA-GFP was used to infect HT22 cells.The infection efficiency was observed under microscope,and the silence efficiency was tested by Real-time PCR and Western Blot.The GLP-1R gene silenced HT22 cells were divided into four groups: the lentivirus group(LV-shGLP-1R group),lentivirus+Aβ31-35group(LV-shGLP-1R+Aβ31-35 group),lentivirus+Oxy+Aβ31-35 group(LV-shGLP-1R+Oxy+Aβ31-35 group)and lentivirus+Oxy group(LV-shGLP-1R+Oxy group).Meanwhile,the uninfected HT22 cells were used as control group.Real-time PCR was used to detect the rhythmic expression of circadian clock genes Bmal1 and Per2 mRNA at CT4,CT8,CT12,CT16,CT20 and CT24 time points respectively.Results:(1)Compared with control group,the expression of Bmal1 and Per2 mRNA in HT22 cells of LV-shGLP-1R group declined at all time points.(2)Compared with LV-shGLP-1R+Aβ31-35 group,there was no obvious improvement of the abnormal expressions of Bmal1 and Per2 mRNA in LV-shGLP-1R+Oxy+Aβ31-35 group.Conclusion:GLP-1R gene silencing decreases the expression of Bmal1 and Per2 mRNA in HT22 cells.After GLP-1R gene silencing,Oxy could not antagonize Aβ31-35 induced abnormal expressions of Bmal1 and Per2 in HT22 cells.