热休克蛋白B 8对低糖诱导小鼠海马神经元自噬的影响

Effect of HSPB8 on Autophagy of Hippocampal Neurons in Low Glucose Induced Mice

目的探讨低糖刺激下小鼠海马神经元热休克蛋白B8(heat shock protein beta-8,HSPB8)和自噬的改变,及HSPB8对自噬的调控作用。方法 HT-22(小鼠海马神经元细胞株)细胞在葡萄糖浓度为2.5和5mmol·L^(-1)DMEM培养基中培养1h后,采用Western blot法检HSPB8,Bcl-2相关永生化基因3(BCL2-Associated Athanogene 3,BAG3)、自噬相关蛋白Beclin-1和微管相关蛋白1-轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达变化;GFP-LC3融合蛋白示踪法观察HT-22细胞低糖(2.5mmol·L^(-1))刺激下的自噬水平;使HT-22细胞过表达HSPB8蛋白后低糖(2.5mmol·L^(-1))刺激1h,Western blot法检测HSPB8,BAG3,LC3-Ⅱ,Beclin-1蛋白表达变化。结果 Western blot结果表明,HT-22细胞低糖(5mmol·L^(-1))刺激1h,HSPB8,BAG3,Beclin-1的表达水平均升高(P均<0.05),而LC3-Ⅱ表达水平无明显变化(P>0.05);低糖(2.5mmol·L^(-1))刺激1h,HSPB8,BAG3,LC3-Ⅱ,Beclin-1表达水平均上调(P<0.05或<0.01);GFP-LC3融合蛋白示踪法显示低糖(2.5mmol·L^(-1))刺激后HT-22细胞绿色荧光斑点增多(P<0.01);过表达HSPB8后低糖(2.5mmol·L^(-1))刺激1h,BAG3,LC3-Ⅱ,Beclin-1表达水平上调(P<0.05或<0.01)。结论小鼠海马神经元在低糖刺激下,HSPB8,BAG3及自噬水平均升高,HSPB8可以促进自噬的发生。

Objective To observe the effect of of Heat shock protein beta-8 （HSPB8） on autophagy of hippocampal neurons in low glucose induced mice. Methods The expression of HSPB8, BCL2-Associated Athanogene 3 （BAG3）, autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3 （LC3） were detected by Western blot after HT-22 （Mouse hippocampal neuronal cell line） cells cultured for 1h in DMEM medium at a glucose concentration of 2.5 and 5 mmol· L-1. GFP-LC3 fusion protein was used to observe the autophagic level of HT-22 cells under low glucose （2.5 mmol· L-1） stimulation. HT-22 cells were stimulated with low glucose （2.5 mmol ·L-1） for lh while overexpressing HSPB8 protein, then the expression of HSPB8, BAG3,LC3- II and Beclin-1 protein was detected by Western blot. Results Western blot analysis showed that the expression levels of HSPB8, BAG3 and Beclin-1 were significantly increased in HT-22 cells cultured for 1 h in DMEM medium at a glucose concentration of 5mmol· L-1 （P〈0.05）, while the expression of LC3- Ⅱ did not change significantly （P〉0.05）. The expression levels of HSPB8, BAG3, LC3- Ⅱ and Beclin-1 were up-regulated in HT-22 cells cultured for lh in DMEM medium at a glucose concentration of 2.5mmol· L-1 （P〈0.05 or 〈0.01 ）, GFP-LC3 fusion protein showed that the green fluorescent spots of HT-22 cells were increased after low glucose （ 2.5mmol· L-1 ） stimulation （ P〈0.01 ） ; After low glucose （ 2.5mmol · L-1 ） stimulation, while overexpressing HSPB8, the expression levels of BAG3, LC3- Ⅱ and Beclin-1 were up-regulated （P〈0.05 or〈0.01）. Conclusion The levels of HSPB8, BAG3 and autophagy were elevated in hippocampal neurons of mice and HSPB8 could promote autophagy.