糖尿病小鼠海马神经元中自噬增强与热休克蛋白B8上调相关

Increased autophagy in hippocampal neurons of diabetic mice is related to up-regulation of heat-shock protein B8

目的探讨糖尿病小鼠海马神经元中热休克蛋白B8(HSPB8)及自噬变化。方法选取8周龄雄性C57BL/KS db/db小鼠10只为糖尿病组,野生型(db/m)小鼠10只为对照组。测定空腹血糖及体重,通过Western blot检测各组海马组织中HSPB8、Bcl-2相关永生化基因3(BAG3)、自噬相关蛋白Beclin-1和微管相关蛋白1-轻链3(LC3)表达。培养HT-22细胞分别用18、25和33 mmol/L的葡萄糖浓度刺激48 h,作为高糖体外模型,Western blot检测各组HSPB8、BAG3、Beclin-1和LC3蛋白表达。应用细胞转染技术分别过表达和沉默HSPB8蛋白后,Western blot检测BAG3,P62和LC3蛋白表达。应用33 mmol/L高糖刺激HT-22细胞,采用mRFP-GFP-LC3工具质粒观察自噬水平。组间比较采用独立样本t检验和单因素方差分析。结果与db/m组相比,db/db小鼠海马组织中HSPB8、BAG3、LC3-Ⅱ/LC3-Ⅰ和Beclin-1表达均升高(1.647±0.150比0.781±0.181、1.832±0.243比0.821±0.197、0.756±0.104比0.518±0.170、1.431±0.201比1.014±0.118,t=4.130~9.131,均P<0.05)。与对照组相比,高糖刺激的HT-22细胞中HSPB8、BAG3、Beclin-1和LC3-Ⅱ/LC3-Ⅰ蛋白表达水平均呈浓度依赖性升高(P<0.05)。较空载组,HSPB8沉默组BAG3、LC3-Ⅱ/LC3-Ⅰ表达降低,P62表达反之,而HSPB8过表达组BAG3、LC3-Ⅱ/LC3-Ⅰ表达水平上调,P62表达下降(0.481±0.161比1.804±0.147、0.615±0.311比2.091±0.210、1.013±0.213比1.615±0.194、1.211±0.154比0.978±0.187;1.817±0.152比0.891±0.151、1.112±0.091比0.743±0.162、1.692±0.170比0.587±0.132、1.236±0.186比1.751±0.131;t=5.560~20.510,均P<0.05)。RFP-GFP-LC3荧光双标质粒显示,较空载组,高糖刺激HSPB8过表达的HT-22细胞中红色荧光斑点增多(2.059±0.310比0.819±0.151,t=9.830,P<0.01)。结论8周龄糖尿病小鼠海马组织中HSPB8表达上调,且可能通过促进自噬体与溶酶体融合,增强自噬。

Objective To investigate the changes of Heat shock protein B8(HSPB8)and autophagy in hippocampal neurons of diabetic mice.Methods Ten 8-week-old male C57BL/KS db/db mice were selected as the diabetes group and 10 wild-type(db/m)mice were selected as the control group.Fasting blood glucose and body weight were measured.Western blot was used to detect the expression of HSPB8,Bcl-2-related immortalized gene 3(BAG3),autophagy related protein beclin-1 and microtubule related protein 1-light chain 3(LC3)in hippocampus of each group.HT-22 cells were cultured and stimulated with glucose concentrations of 18,25 and 33 mmol/L for 48 h respectively,as high glucose models in vitro.The HSPB8,BAG3,beclin-1 and LC3 proteins in each group were detected by Western blot.The expression of BAG3,P62 and LC3 was detected by Western blot after overexpression and silencing of HSPB8 by cell transfection.Ht-22 cells were stimulated by 33 mmol/L high glucose,and the autophagy was observed by RFP-GFP-LC3.Independent sample t test and one way analysis of variance were used for statistical analysis.Results Compared with db/m group,the expression of HSPB8,BAG3,Beclin-1 and LC3-Ⅱ/LC3-Ⅰin the hippocampus of db/db mice increased(1.647±0.150 vs 0.781±0.181,1.832±0.243 vs 0.821±0.197,0.756±0.104 vs 0.518±0.170,1.431±0.201 vs 1.014±0.118,t=4.130-9.131,all P<0.05).Compared with the control group,the expression levels of HSPB8,BAG3,Beclin-1 and LC3-Ⅱ/LC3-Ⅰprotein in high glucose stimulated HT-22 cells increased in a concentration dependent manner(all P<0.05).Compared with the control group,the expression of BAG3,LC3-Ⅱ/LC3-Ⅰdecreased in the HSPB8 knocked group,while p62 expression decreased in the HspB8 over expression group(0.481±0.161 vs 1.804±0.147,0.615±0.311 vs 2.091±0.210,1.013±0.213 vs 1.615±0.194,1.211±0.154 vs 0.978±0.187;1.817±0.152 vs 0.891±0.151,1.112±0.091 vs 0.743±0.162,1.692±0.170 vs 0.587±0.132,1.236±0.186 vs 1.751±0.131,t=5.560-20.510,all P<0.05).Compared with the control group,the red fluorescent spots in HSPB8 overexpressed HT-22 cells increased(2.059±0.310 vs 0.819±0.151,t=9.830,P<0.01).Conclusion The expression of HSPB8 in the hippocampal tissue of 8-week-old diabetic mice is up-regulated,which may enhance autophagy by promoting the fusion of autophagosome and lysosome.