糖原合成酶激酶3β活性增加参与β淀粉样蛋白31~35诱导的HT22海马神经细胞Bmal1表达降低  

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells

作　　者：郭帅 孙聪 王昌图 原媛 宁娜 侯晓鸿 王丽[1] 王晓晖[1] Guo Shuai;Sun Cong;Wang Changtu;Yuan Yuan;Ning Na;Hou Xiaohong;Wang Li;Wang Xiaohui(Department of Pathology,Shanxi Medical University,Taiyuan 030001,China;Department of Morphology,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院病理教研室,太原030001 [2]山西医科大学基础医学院形态学实验室,太原030001

出　　处：《中华神经科杂志》2020年第2期96-102,共7页Chinese Journal of Neurology

基　　金：国家自然科学基金资助项目(81471343);2018年度山西省留学人员科技活动项目重点项目(王晓晖)。

摘　　要：目的探讨糖原合成酶激酶3β(GSK3β)在β淀粉样蛋白31~35(Aβ31~35)引起小鼠海马HT22神经细胞Bmal1基因/蛋白表达降低中的作用。方法采用小鼠海马神经细胞HT22作为实验对象,将HT22细胞按随机数字表法分为对照组、Aβ31~35处理组、LiCl+Aβ31~35处理组。以1%血清饥饿1 h来诱导细胞同步化(昼夜时间0,即CT0)。采用细胞增殖-毒性检测法检测细胞存活率;采用实时荧光定量PCR法检测上述各组细胞CT4、CT8、CT12、CT16、CT20、CT24时间点Bmal1基因的表达水平;采用Western印迹方法检测各组GSK3β表达情况及BMAL1蛋白表达水平。结果Aβ31~35诱导HT22细胞Bmal1 mRNA及BMAL1蛋白水平在CT20时间点较对照组显著降低(Bmal1 mRNA:分别为0.38±0.06与0.83±0.08,t=4.549,P=0.001;BMAL1蛋白:分别为0.67±0.04与1.00±0.04,t=5.943,P<0.001)。与对照组相比,Aβ31~35引起HT22细胞GSK3β活性增加,表现为GSK3βSer9位点(GSK3βS9)磷酸化水平较对照组显著降低,磷酸化GSK3βS9与GSK3β比值下降(分别为0.66±0.08与1.02±0.14,t=2.217,P=0.025);Aβ31~35引起HT22细胞存活率显著降低(分别为71.85%±6.20%与98.14%±2.68%,t=3.891,P=0.006),GSK3β抑制剂LiCl预处理后有效逆转Aβ31~35诱导的HT22细胞存活率下降(LiCl+Aβ31~35处理组与Aβ31~35处理组分别为90.74%±5.74%与71.85%±6.20%,t=3.412,P=0.010);LiCl预处理可以明显逆转Aβ31~35所致CT20时间点Bmal1 mRNA及BMAL1蛋白水平降低(LiCl+Aβ31~35处理组与Aβ31~35处理组Bmal1 mRNA分别为:0.72±0.05与0.38±0.06,t=4.378,P=0.001;BMAL1蛋白分别为:0.90±0.04与0.67±0.04,t=4.052,P=0.002)。结论GSK3β活性增加参与Aβ31~35引起的HT22细胞Bmal1基因/蛋白表达降低。Objective To investigate the effect of glycogen synthase kinase 3β(GSK3β)on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35(Aβ31-35)in HT22 cells.Methods HT22 mouse hippocampal cells were divided into control group,Aβ31-35 group and LiCl+Aβ31-35 group by random number table method in the present study.Cells were synchronized to G0/G1 phase by 1%serum starvation for 1 hour(circadian time 0(CT0)).Cell viability was detected by the cell counting kit-8 assay.The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times.The expression of GSK3βand BMAL1 protein was detected by Western blotting.Results Compared with the control group,Aβ31-35 induced the decreased expression of Bmal1 mRNA;The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20(Bmal1 mRNA:0.38±0.06 vs 0.83±0.08,t=4.549,P=0.001;BMAL1 protein:0.67±0.04 vs 1.00±0.04,t=5.943,P<0.001).In the Aβ31-35 group,GSK3βactivity was increased and the ratio of phosphorylated GSK3βS9 to GSK3βwas decreased compared to the control group(0.66±0.08 vs 1.02±0.14,t=2.217,P=0.025).Aβ31-35 decreased the viability of HT22 cells(71.85%±6.20%in the Aβ31-35 group vs 98.14%±2.68%in the control group,t=3.891,P=0.006),and the GSK3βinhibitor LiCl pretreatment effectively reversed the decline of the viability induced by Aβ31-35(90.74%±5.74%in the LiCl+Aβ31-35 group vs 71.85%±6.20%in the Aβ31-35 group,t=3.412,P=0.010).LiCl(in the LiCl+Aβ31-35 group)increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group(Bmal1 mRNA:0.72±0.05 vs 0.38±0.06,t=4.378,P=0.001;BMAL1 protein:0.90±0.04 vs 0.67±0.04,t=4.052,P=0.002).Conclusion Increased GSK3βactivity involved in the decreased expression of Bmal1 induced by Aβ31-35 in HT22 cells.

关 键 词：淀粉样蛋白 糖原合成酶激酶3 Bmal1基因 HT22细胞 

分 类 号：R741.02[医药卫生—神经病学与精神病学]