| Alzheimer’s disease(AD) is a familiar neurodegenerative disease, characterized by deteriorating dysfunction of cognitive, learning and memory ability. Further studies indicated that 25~40% AD patients suffered from different degrees of circadian rhythm disorders, including sleep-wake cycle disorder, Sundowning, excessive daytime sleepiness and bedtime resistance, which not only influence the cognitive behavior dysfunction, but also diminish quality of life. The major pathological characteristics of AD are deposition of senile plaques in neuronal gap, neurofibrillary tangles, loss of neurons. Amyloid β-protein(Aβ) is main constituent of senile plaques, displaying distinct neurotoxic effect. A study found significant circadian rhythm disorders in transgenic mice with five familial AD(5×FAD). However, whether Aβ can induce circadian rhythm disorders in normal mice is still unknown.Several researches found close relationships between type II diabetes mellitus(T2DM)and AD in some respects of pathogeny and pathogenesis process. Surprisingly, Exendin-4,a new therapeutic drug for T2 DM, exerts neuroprotective effect. However, the improvement effect of Exendin-4 on circadian rhythm disorders is not clear.Therefore, the objective of this study are:(1) Observe the effects of Aβ31-35 on circadian rhythm in C57BL/6 mice, and the expression of core circadian protein CLOCK in hippocampus.(2) Observe the effects of Exendin-4 on the circadian rhythm disorders induced by Aβ31-35 in C57BL/6 mice, and the expression of CLOCK in hippocampus,aiming at explore the neuroprotective effect of Exendin-4.Part I Aβ31-35 leads to circadian rhythm disorders and abnormal expression of CLOCK protein in C57BL/6 miceObjective:Running wheel activity was observed to mirror the changes in circadian rhythm induced by Aβ31-35, and the expression of CLOCK protein in hippocampus was detectedby Western blot.Methods:Healthy male C57BL/6 mice were randomly divided into Control group and Aβ31-35 group, Control mice were injected with 8.2 μL triple-distilled water into bilateral hippocampal, and Aβ31-35 group were injected with 8.2 μL Aβ31-35(15 nmol) into bilateral hippocampus. The mice were placed in 12hr/12 hr light-dark cycle(light-dark cycle, LD) for the first week, then the lighting condition was changed into constant darkness(dark-dark, DD). Using Vital View software to record the running wheel activity of C57BL/6 mice in each group. Experimental dates were exported, then the free-running period, amplitude, rhythm robustness and locomotive activity of each mouse in DD were analysed by Acti View software.Under dark condition, the mice were sacrificed at different circadian times(CTs),removing the hippocampus. After adding tissue lysate, the hippocampus were minced and homogenized on ice, and then centrifuged and got supernatant, using BCA method to measure protein concentration, calculated samples volume. Electrophoresis, transferred to PVDF membrane, blocked, incubated with primary antibody, washed the membrane,incubated with secondary antibody, exposed. Band grayscale values were measured by Image J software, calculating CLOCK protein relative expression at different CTs.Results:In Control group, mice displayed stable circadian rhythm in activity, distinct dividing line in resting phase and active phase, and the activity were observed during subjective night. While, the circadian rhythm of mice in Aβ 31-35 group, unstable circadian rhythm,indefinite dividing line between resting phase and active phase, and the activity were increased during subjective day and reduced during subjective night. In Control group, the free running period was 23.51±0.05 hr, the amplitude was 127±14.06 turns, and the robustness was 54.71%±2.55%. While in Aβ 31-35 group, the the free running period was23.63±0.05 hr, the amplitude was 90.44±18.85 turns, and the robustness was22.47%±4.28%, suggesting longer free running period(P<0.05), reduced amplitude(P<0.05) and decreased stability(P<0.05).The expression of CLOCK protein in Control mice show obvious rhythm, including higher expression at CT8 and CT20, and lower expression at CT2 and CT14. After giving Aβ31-35, the expression CLOCK protein is lack of circadian rhythm, lacking of peaks and valleys, and there is a significant difference between Control and Aβ group(P<0.05).Conclusion:Hippocampal injection with Aβ31-35 lead to circadian rhythm disorders in C57BL/6mice, which suggested that Aβ31-35 played a toxic effect on nervous system and disrupted circadian rhythm. Further study showed that Aβ31-35 induced abnormal expression of circadian protein CLOCK in hippocampus, which revealed that the neurotoxicity of Aβ31-35 may be owing to altering CLOCK protein expression.Part II Aβ31-35 leads to circadian rhythm disorders and abnormal expression of CLOCK protein in C57BL/6 miceObjective:The effect of Exendin-4 on circadian rhythm disorders induced Aβ31-35 in C57BL/6mice were observed by wheel running activity; meanwhile, from the perspective of rhythm protein expression, we detected the effect of Exendin-4 on the nervous system.Methods:The C57BL/6 mice were randomly divided into Exendin-4 pretreatment group and Exendin-4 group, Exendin-4 pretreatment mice were injected with 1.6 μL(50 pmol)Exendin-4 into bilateral hippocampal after 15 min injecting with 8.2 μL(15 nmol)Aβ31-35, Exendin-4 group group were injected with 1.6 μL(50 pmol) Exendin-4 after 15 min injecting with 8.2 μL(15 nmol) triple-distilled water into bilateral hippocampus. The mice were placed in LD for the first week, then the lighting condition was changed intoDD for 2 weeks. Then analyzed the free-running period, amplitude, rhythm robustness and locomotive activity of each mouse in DD by Acti View software.After 2 weeks in DD, the mice were killed and the hippocampus were taken out at different CTs, the tissue protein were extracted by tissue lysis to determine the expression of CLOCK protein in hippocampus.Results:After the pre-hippocampal administration of Exendin-4, the wheel running activity of C57BL/6 mice displayed circadian rhythm, including there was a significant dividing line in resting phase and active phase and running was concentrated at the subjective night. In Exendin-4 pretreatment group, the free running period was 23.57±0.07 hr, the amplitude was 125±19.43 turns, and the robustness was 49.34%±3.76%, suggesting shorter free running period(P<0.05), increased amplitude(P<0.05) and improved stability(P<0.05)than in Aβ31-35 group, but that were not different from Control groups(P>0.05).Compared with the Aβ31-35 group, The rhythmic expression of CLOCK protein in Exendin-4 pretreatment group partially recovered, including increased expression at CT8 and CT20, and reduced expression at CT2(P<0.05). Administration alone with Exendin-4didn’t change the normal rhythmic expression of CLOCK protein in the hippocampus(P>0.05).Conclusion:Hippocampal pre-injection of Exendin-4 can partially improve the circadian rhythm disorders induced by Aβ31-35, indicating Exendin-4 can partially antagonize the neurotoxic effect induced by Aβ31-35. Further study indicated the neuroprotective effect of Exendin-4 can be realized through regulating the expression of CLOCK protein. |
PDF Full Text Request