GSK-3β在斑马鱼脑缺氧/复氧损伤中的作用及其对微管相关蛋白2的影响  

The role of GSK-3βin Zebrafish cerebral hypoxia/reoxygenation injury and its effect on microtubule-associated protein 2

作　　者：杨梦思 张丽 胡宪文 Yang Mengsi;Zhang Li;Hu Xianwen(Key Lab of Anesthesiology and Perioperative Medicine of Anhui Higher Education Institutes,Hefei 230601;Dept of Anesthesiology and Perioperative Medicine,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601)

机构地区：[1]麻醉与围术期医学安徽普通高校重点实验室,合肥230601 [2]安徽医科大学第二附属医院麻醉与围术期医学科,合肥230601

出　　处：《安徽医科大学学报》2023年第2期202-208,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:1908085QH358);安徽省卫生健康委科研项目(编号:AHWJ2021a017);安徽医科大学第二附属医院国家自然科学基金孵育计划(编号:2020GQFY01)。

摘　　要：目的探究斑马鱼脑缺氧/复氧(H/R)过程中糖原合酶激酶3β(GSK-3β)的作用及其对微管相关蛋白2(MAP2)的影响。方法建立斑马鱼H/R模型,选用健康同等体型成鱼,分为对照(Control)组、缺氧/复氧(H/R)组、缺氧/复氧+GSK-3β抑制剂(H/R+TDZD-8)组进行实验。选取各组斑马鱼脑组织,通过qRT-PCR测定缺氧诱导因子Hif-1αa、Hif-1αb在不同复氧时间点mRNA表达情况,TTC染色与TUNEL染色检测脑梗死面积及细胞凋亡,Western blot检测Hif-1α、GSK-3β、p-GSK-3β(Ser 9)、MAP2的蛋白表达水平,免疫荧光染色检测MAP2在脑中的分布和表达情况。结果与Control组相比,H/R组Hif-1αa、Hif-1αb的mRNA及Hif-1α蛋白表达水平升高(P<0.01),脑梗死面积及凋亡细胞增多(P<0.01),p-GSK-3β(Ser 9)/GSK-3β比值、MAP2蛋白表达降低(P<0.05),MAP2免疫荧光表达下降(P<0.01);与H/R组比较,TDZD-8预处理能够减少斑马鱼脑梗死面积及细胞凋亡(P<0.01),增加p-GSK-3β(Ser 9)/GSK-3β比值、MAP2蛋白表达(P<0.01),增加MAP2免疫荧光表达(P<0.01)。结论缺氧/复氧可造成斑马鱼脑神经元损伤,其机制可能与抑制GSK-3β磷酸化和MAP2的表达有关。GSK-3β特异性抑制剂TDZD-8可通过促进p-GSK-3β(Ser 9)的表达,减少MAP2的降解,从而逆转缺氧/复氧对斑马鱼脑神经元的损伤。Objective To investigate the effect of glycogen synthase kinase 3β(GSK-3β)and its correlation with microtubule-associated protein 2(MAP2)during cerebral hypoxia/reoxygenation(H/R)in zebrafish.Methods The cerebral hypoxia/reoxygenation model of zebrafish was established.Healthy adult zebrafishes of the same size were divided into control group(Control),hypoxia/reoxygenation group(H/R)and hypoxia/reoxygenation+GSK-3β inhibitor group(H/R+TDZD-8)for experiment.The brain tissues of zebrafish in each group were selected to determine the mRNA expressions of hypoxia inducible factor 1αa and 1αb(Hi F-1αa and HIF-1αb)at different reoxygenation time points by qRT-PCR,and the protein expression levels of HIF-1α,GSK-3β,p-GSK-3β(Ser9)and MAP2 were detected by Western blot,TTC staining and TUNEL staining were used to detect cerebral infarction area and cell apoptosis,and immunofluorescence was used to detect the distribution and expression of MAP2 in brain.Results Compared with Control group,the mRNA levels of Hif-1αa and Hif-1αb(P<0.01)and protein expression of Hif-1α(P<0.01)increased in H/R group,the area of cerebral infarction(P<0.01)and apoptotic cells(P<0.01)increased,p-GSK-3β(Ser 9)/GSK-3β ratio,MAP2 protein expression(P<0.05)and immunofluorescence expression of MAP2(P<0.01)reduced;Furthermore,TDZD-8 pretreatment could relieve the brain injury of H/R zebrafish by decreasing the infarct size and cell apoptosis,improving the ratio of pGSK-3β(Ser 9)/GSK-3β,and increasing the expression of MAP2.Conclusion Hypoxia/reoxygenation can cause brain neuron damage in zebrafish,and its mechanism may be related to inhibition of GSK-3βphosphorylation and MAP2 expression.GSK-3β specific inhibitor TDZD-8 can reverse the damage of brain neurons caused by hypoxia/reoxygenation by promoting the expression of P-GSK-3β(Ser 9)and reducing MAP2 degradation.

关 键 词：斑马鱼 脑缺氧/复氧损伤 糖原合酶激酶3Β 微管相关蛋白2 TDZD-8 

分 类 号：R332[医药卫生—人体生理学]