DA-JC1改善β-淀粉样蛋白31-35所致昼夜节律紊乱研究

DA-JC1 improves circadian rhythm disorder induced by amyloid β-protein 31-35

目的 观察新型胰高血糖素受体/葡萄糖依赖性促胰岛素受体双受体激动剂DA-JC1对β-淀粉样蛋白31-35（Aβ31-35）引起的C57BL/6小鼠昼夜节律紊乱以及小鼠海马HT22神经细胞period1基因/蛋白表达异常的影响.方法 （1）选取6-8周龄雄性C57BL/6小鼠进行跑轮行为学实验,分析DA-JC1在改善Aβ31-35所致小鼠昼夜节律紊乱中的作用.（2）选取HT22神经细胞,采用随机数字表法将其分为对照组、Aβ31-35组、DA-JC1预处理组、DA-JC1组（各组n=4）,采用四甲基偶氮唑盐法检测各组细胞的存活率.采用实时荧光定量PCR方法检测各昼夜时间的时间点period1基因表达水平,通过蛋白质印迹方法检测period1差异最大昼夜时间的period1蛋白表达水平,观察DA-JC1对Aβ31-35引起的小鼠海马HT22神经细胞period1表达异常的影响.结果 （1）与对照组相比,Aβ31-35引起小鼠自由运转周期显著延长[（23.80±0.06）h与（23.54 ±0.07）h,t=0.265,P=0.010],DA-JC1预处理后显著缩短了Aβ31-35引起的自由运转周期延长[（23.61 ±0.06）h,t=0.193,P=0.047].（2）与对照组相比,5μmol/L Aβ31-35可引起HT22细胞存活率显著降低（78.7％±3.4％与100.0％±3.6％,t=12.393,P=0.005）,而DA-JC1预处理1h后细胞存活率明显回升（89.2％±2.3％,=9.748,P=0.048）.（3） Aβ31-35导致HT22细胞period1表达异常,表现在昼夜时间12时（period1基因：0.58±0.04;period1蛋白：0.74±0.07）较对照组（period1基因：1.00±0.09,=0.419,P=0.001;period1蛋白：1.01±0.07,t=0.221,P=0.007）表达显著降低,DA-JC1可以逆转Aβ31-35在昼夜时间12时诱导的HT22细胞period1表达水平降低（period1基因：0.79 ±0.11,t=0.279,P=0.024;penod1蛋白：0.99±0.05,t=0.226,P=0.009）.结论 DA-JC1可以有效缓解Aβ31-35所致C57BL/6小鼠昼夜节律紊乱,同时可以有效改善Aβ31-35所致HT22细胞中period1表达异常.

Objective To observe the effect of DA-JC1 on the circadian rhythm disorder in C57BL/6 mice and the abnormal expression of period1 in HT22 cells induced by amyloid β-protein 31-35 （Aβ31-35）.Methods （1） The six-eight weeks old C57BL/6 male mice were selected for wheelrunning behavior experiment.Then we analyzed the effect of DA-JC1 on the circadian rhythm disorder induced by Aβ31-35.（2） HT22 mouse hippocampal cells were adopted as the research objects.Cells were divided into vehicle group,Aβ31-35 group,pre-DA-JC1 group and DA-JC1 group （n =4 respectively） by random number table method.Cell viability was assessed by methylthiazolyldiphenyl-tetrazolium bromide cytotoxicity assay.Real-time quantitative PCR was used to detect the expression of clock gene period1,and Western blotting was applied to examine the expression of period1 protein at circadian time （CT） 12.Results （1） Compared with the vehicle group （（23.54 ± 0.07） h）,the circadian rhythm of mice in the Aβ31-35 group was disturbed which exhibited significantly longer free running period （（23.80 ± 0.06） h,t=0.265,P=0.010）,whereas the disruption was significantly relieved with pre-treatment of DA-JC1 （（23.61 ± 0.06） h,t =0.193,P =0.047）.（2） Compared with the vehicle group （100.0% ± 3.6%）,5 μmol/L Aβ31-35 decreased the cell viability significantly （78.7% ± 3.4%,t =12.393,P =0.005）,and DA-JC1 can reduce the toxicity of Aβ31-35 in HT22 cells （89.2% ± 2.3%,t =9.748,P =0.048）.（3） Compared with the vehicle group （period1 gene：1.00 ± 0.09;period1 protein：1.01 ± 0.07）,abnormal rhythmic expression of period1 was induced by Aβ31-35 in HT22 cells which significantly decreased at CT12 （period1 gene：0.58 ± 0.04,t =0.419,P =0.001;period1 protein：0.74 ± 0.07,t =0.221,P =0.007） while DA-JC1 pre-treatment can reverse the abnormal expression （period1 gene：0.79 ±0.11,t =0.279,P=0.024;period1 protein：0.99 ±0.05,t=0.226,P=0.009）.Conclusion DA-JC1 improves the circadian rhythm disorder induced by Aβ31-35 in C57BL/6 mice and improves the abnormal expression of period1 induced by Aβ31-35 in HT22 cells.