抑制大鼠脑缺血再灌注损伤中GSK-3β活性对自噬的影响及可能机制

Influence of glycogen synthase kinase-3β inhibition on autophagy in rats with cerebral ischemia/reperfusion injury and possible mechanism

摘要:

目的：探讨大鼠缺血再灌注损伤中抑制糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK-3β）后对自噬的影响及如何通过酵母自噬启动ATG1激酶同源蛋白（Unc-51 like autophagy activating kinase 1，ULK1）影响自噬的机制。方法：实验选取SD大鼠为研究对象，分为假手术组（Sham 组，仅结扎颈内动脉）、缺血再灌注组（MCAO 组，大脑中动脉栓塞术制备局灶脑缺血再灌注模型）、GSK-3β干扰片段组（siGSK-3β组，建模前24 h注射GSK-3β干扰片段）、GSK-3β无意义序列组（ConsiGSK-3β组，建模前24 h注射无意义序列）、GSK-3β抑制剂组（SB216763组，建模前6 h注射抑制剂SB216763），每组5只。免疫印迹检测大脑皮质酪氨酸216号位点磷酸化GSK-3β[GSK-3β（P-tyr216）]、自噬相关蛋白轻链3抗体Ⅰ/Ⅱ（autophagy microtubule-as－sociated protein light chain 3 antibodyⅠ/Ⅱ，LC3BⅠ/Ⅱ）、泛素结合蛋白（sequestosome 1，P62）、磷酸化ULK1（phosphorylated ULK1，P-ULK1）、乙酰化ULK1（acetylated ULK1，AcK）的表达，电镜显示自噬体数量。结果：与Sham组比较，MCAO组中GSK-3β（P-tyr216）增高（P=0.000）。siGSK-3β、SB216763组相较MCAO组，LC3BⅡ/LC3BⅠ升高（P=0.000）、P62表达下降（P=0.000）、P-ULK1表达增高（P=0.000）、AcK表达下降（SB216763：P＜0.05；siGSK-3β：P＜0.01），电镜可见自噬小体。结论：在脑缺血再灌注损伤中，GSK-3β活性抑制后可通过磷酸化ULK1增强细胞自噬。

Abstract:

Objective：To investigate the influence of glycogen synthase kinase-3β（GSK-3β） inhibition on autophagy in rats with cere－bral ischemia/reperfusion injury and the mechanism of affecting autophagy via Unc-51 like autophagy activating kinase 1（ULK1）. Methods：Sprague-Dawley rats were divided into sham-operation group（Sham group，ligation of the internal carotid artery alone），middle cerebral artery occlusion（MCAO） group（MCAO was performed to establish a model of focal cerebral ischemia/reperfusion），GSK-3β interference fragment group（siGSK-3β group；the GSK-3β interference fragment was injected at 24 hours before model establishment），GSK-3β non-significant sequence group（ConsiGSK-3β group；non-significant sequence was injected at 24 hours before model establishment），and GSK-3β inhibitor group（SB216763 group；the inhibitor SB216763 was injected at 6 hours before model establishment），with 5 rats in each group. Western blotting was used to measure the expression of tyrosine-216-phosphorylated GSK-3β [GSK-3β（P-tyr216）]，autophagy microtubule-associated protein light chain 3 antibody Ⅰ/Ⅱ（LC3B Ⅰ/Ⅱ），sequestosome 1（P62），phosphorylated ULK1（P-ULK1），and acetylated ULK1 （AcK） in the cerebral cortex. An electron microscope was used to measure the number of autophagosomes. Results：Compared with the Sham group，the MCAO group had a significant increase in the expression of GSK-3β（P-tyr216）（P=0.000）. Compared with the MCAO group，the siGSK-3β group and the SB216763 group had significant increases in the expression of LC3B I/II and P-ULK1（P=0.000） and significant reductions in the expression of P62（P=0.000） and AcK（SB216763 group：P<0.05；siGSK-3β group：P<0.01），and autophagosomes were observed under the electron microscope. Conclusion：GSK-3β inhibition can enhance au－tophagy via phosphorylated ULK1 in cerebral ischemia/reperfu－sion injury.