| Abnormal accumulation of amyloid β-protein in the brain is the pathological basis of circadian rhythm disorder in Alzheimer’s disease.It was known that neurons are extremely sensitive to changes in intracellular level of Ca2+,and it has been foundthat Aβ deposition in the brains of double-transgenic mice,where the calcium steady-state is imbalance.And the normal function of the nervous system requires the participation of calcium signaling.The study found that periodic calcium influx is an important reason to maintain SCN rhythm.It can be seen that calcium signal is closely related to the formation and maintenance of circadian rhythm.Per1,as a major output factor of circadian system,plays an important role in regulating circadian rhythm cycle and maintaining circadian rhythm stability.It was found that the expression of per1 gene can be affected by the change of calcium level.The effect of Aβ-induced calcium overload on Per1 expression has not been reported.The objective of this study was to investigate the effect of Aβ31-35-induced calcium overload on Per1 expression at the cellular level.Exendin-4,a long-acting agonist of the hormone glucagon-like peptide 1 receptor,shows a neuroprotective effect in many studies.Our previous study identified that Exendin-4 plays an important role in regulating circadian rhythm disorders,but the mechanism of action are still unclear.In this study,the hippocampal neuronal cell line HT22 was used as the experimental object.The expression of per1 m RNA in different treatment groups was detected at each time point,and the relative expression of PER1 protein,its expression in situ and intracellular Ca2+ levels were detected at the most significant CT time to further explore the effect of Aβ31-35 on Per1 expression and the possible mechanism of neuroprotection by Exendin-4.Part I: Aβ31-35 leads to abnormal expression of Per1 in HT22 cells Object:To observe the effect of Aβ31-35 on the survival rate,morphology and Per1 expression in HT22 cells.Method:The cultured cells were randomly divided into control group and Aβ31-35 group,then 1% FBS starvation for 1 h to induce cell synchronization.After the cells were synchronized,the control group was further cultured with complete medium,while the Aβ31-35 group was replaced with a final concentration of 25 μM Aβ31-35.MTT assay was used to observe the cell viability.The cell morphology was imaged with an inverted microscope.The expression of Per1 mRNA was quantified by Real-time PCR in different CT time.And Western blot was employed to detect the expression of PER1 protein in the most significant CT time,while its expression in situ was detected by immunofluorescence staining.Results:The cell survival rate was significantly lower(63±2.34%)than that of the control group(100±0.66%)after 25 μM Aβ31-35 for 24 h.After exposure to Aβ31-35 for 24 h,the number of cells was less than that of the control group,the cell body shrinks,the number of round cells increased and the cell morphology was abnormal.Aβ31-35 affected the expression of per1 gene and the expression of per1 mRNA(1.43±0.18)was significantly higher than that of the control group(0.65 ± 0.11)at the time of CT16(p<0.05).The expression of the clock protein PER1 was detected at the time point of this action time.Compared with the control group(1.0±0.07)the relative expression of PER1 protein after treatment with Aβ31-35 was(0.59±0.01),and also the fluorescence intensity of PER1 protein was significantly decreased(51.76±2.52%,p<0.05)after Aβ31-35 treated,and the difference was statistically significant.Conclusion:Aβ31-35 induced a decrease in viability,and abnormal cell morphology and abnormal Per1 expression.PartⅡ: Exendin-4 improves the abnormal of Per1 induced by Aβ31-35Object:Observe the protective effect of Exendin-4 pretreatment for 1 h on Aβ31-35 induced decreased cell survival rate,morphological changes,Per1 abnormal expression and its possible mechanism.Method:MTT assay was used to detect cell viability.Real-time PCR was used to detect the expression of per1 mRNA in each CT time after 1 h of pretend treated with Exendin-4.Western blot was used to detect the relative expression of PER1 protein after pretreatment with Exendin-4 in the most significant CT time.And the expression of PER1 protein in situ was detected by immunofluorescence staining in the most significant CT time.Result:Pretreatment with Exendin-4 significantly improved(77±2.68%)Aβ31-35 induced decreased cell viability,and we found that the damaged cell morphology can be partially restored.Exendin-4 pretreatment for 1 h could partially improve the abnormal expression of per1 gene and significantly decrease the expression of per1gene(0.77±0.17,p<0.05)caused by Aβ31-35 at CT16.Compared with Aβ31-35 group,the relative expression of PER1 protein was significantly increased by Exendin-4 pretreatment(0.80±0.03).The fluorescence intensity of the protein was also significantly increased(81.18±4.98%,p<0.05).Conclusion:Exendin-4 can improve the survival rate,abnormal morphology and abnormal expression of Per1 caused by Aβ31-35 in HT22 cells.Part Ⅲ: Calcium ion regulation mechanism of Exendin 4 in abnormal expression of Per1 induced by Aβ31-35 in HT22 cells.Object:To investigate the possible mechanism of Aβ31-35 induced abnormal expression of Per1 and the protection of Exendin-4.Method:Real-time PCR was used to detect the expression of per1 gene in different CT time after pretreatment with Nimodipine for 1 h.At the most significant time in the expression of per1,flow cytometry and calcium ion staining were used to detect the changes of intracellular Ca2+ level,which was reflected by the value of the cell fluorescence intensity.Results:Nimodipine pretreatment can play similar roles with Exendin-4,which can improve Aβ31-35 induced per1 abnormal expression.At CT16,Nimodipine significantly reduced Aβ31-35 induced abnormal high expression of per1 gene levels(0.89±0.07,p<0.05).Further detecting the fluorescence intensity of Per1 protein in cells at the time,the fluorescence intensity of Per1 in Aβ31-35 group was(584±31.35),which was significantly higher than that in control group(356±14.97).With Exendin-4 pretreatment,the fluorescence intensity of Per1 was significantly decreased(360±26.81).The data demonstrated that Exendin-4 pretreatment has the same effect with Nimodipine pretreatment,which can reduce the abnormal enhancement of fluorescence intensity(446±13.89).Conclusion:Exendin-4 improves Aβ31-35 induced abnormal expression of Per1 byantagonizing calcium overload in HT22 cells. |
PDF Full Text Request