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(phosphorylatedULK1,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。
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 autophagy via phosphorylated ULK1 in cerebral ischemia/reperfusion injury.
作者
汪悦婷
张鑫
赵敬
Wang Yueting;Zhang Xin;Zhao Jing(Teaching and Research Section of Pathophysiology,College of Basic Medicine,Chongqing Medical University;Department of General Medicine,The Second Affiliated Hospital of Chongqing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第5期551-554,共4页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81671158、81771261)
国家自然科学青年基金资助项目(编号:81701165)
