棕榈酰转移酶ZDHHC3对脑缺血-再灌注损伤的影响  

Effect of palmitoyltransferase ZDHHC3 on cerebral ischemia-reperfusion injury

作　　者：王萱 陈莎 邓玲[1] 刘京东 赵虹霞 刘胜伟 董志[1] WANG Xuan;CHEN Sha;DENG Ling;LIU Jing-dong;ZHAO Hong-xia;LIU Shengwei;DONG Zhi(Department of Pharmacology,Pharmacy School of Chongqing Medical University,Chongqing Key Laboratory of Bio-chemistry and Molecular Pharmacology,Chongqing 400016,China)

机构地区：[1]重庆医科大学药理学教研室,重庆市生物化学与分子药理学重点实验室,重庆400016

出　　处：《中国病理生理杂志》2021年第5期789-797,共9页Chinese Journal of Pathophysiology

基　　金：重庆市自然科学基金资助项目(No.cstc2018jcyjAX0378)。

摘　　要：目的:探讨棕榈酰转移酶ZDHHC3在脑缺血-再灌注损伤大鼠及氧糖剥夺/复氧(OGD/R)细胞模型中的表达和保护作用。方法:制备大脑中动脉闭塞(MCAO)的SD大鼠模型,缺血90 min,再灌注24 h后,RT-qPCR和Western blot测定大鼠大脑皮层和海马组织中ZDHHC3的mRNA和蛋白水平。免疫荧光双标测定ZDHHC3在大鼠脑中的细胞定位。建立OGD/R的PC12细胞模型,RT-qPCR和Western blot测定PC12细胞中ZDHHC3的mRNA和蛋白表达水平。在OGD/R模型中,转染ZDHHC3过表达质粒,Western blot检测ZDHHC3蛋白的表达情况及过表达ZDHHC3后突触相关蛋白突触后致密蛋白95(PSD95)和AMPA受体2(AMPAR2)及凋亡相关蛋白Bcl-2和Bax的表达水平,MTT法检测细胞活力,流式细胞术分析细胞凋亡程度。结果:与对照组相比,ZDHHC3和PSD95在MCAO大鼠大脑皮层和海马及OGD/R损伤的PC12细胞中的mRNA和蛋白表达水平降低(P<0.05)。ZDHHC3在大鼠神经元中表达,但在星形胶质细胞中不表达。与模型组相比,过表达ZDHHC3的细胞中PSD95和AMPAR2表达增加,Bcl-2表达增加,Bax表达下降(P<0.01),细胞活力增强,细胞凋亡率减少(P<0.05)。结论:ZDHHC3可能通过激活突触相关蛋白,调节突触可塑性,减少细胞凋亡,从而对脑缺血-再灌注损伤大鼠发挥保护作用。AIM:To investigate the expression of palmitoyltransferase ZDHHC3 in the brain of rats with cere-bral ischemia-reperfusion injury(CIRI)and its effect on oxygen-glucose deprivation/reoxygenation(OGD/R)cell model.METHODS:Middle cerebral artery occlusion(MCAO)was used to establish the model of CIRI.After ischemia for 90 min and reperfusion for 24 h,Western blot and RT-qPCR were used to determine the expression of ZDHHC3 at protein and mRNA levels in MCAO rats,and the cellular distribution of ZDHHC3 in rats was determined by double-labeled immu-nofluorescence technique.The PC12 cell model of OGD/R was established to determine the mRNA and protein levels of ZDHHC3 by RT-qPCR and Western blot.The PC12 cells were transfected with pcDNA3.1-ZDHHC3,and the effects of ZDHHC3 over-expression on cell viability and apoptosis were observed by MTT assay and flow cytometry.The protein ex-pression of postsynaptic density protein 95(PSD95),AMPA receptor 2(AMPAR2),Bcl-2 and Bax was determined by Western blot.RESULTS:Decreased expression of ZDHHC3 in the cortex and hippocampus of MCAO rats and OGD/Rtreated PC12 cells was observed as compared with control groups(P<0.05).The ZDHHC3 expression was identified in the cytomembranes and cytoplasm of neurons in the hippocampus and cortex of MCAO rats,but not in astrocytes.In PC12 cells,compared with model group and pcDNA3.1-GFP group,the expression levels of synapse-related proteins PSD95 and AMPAR2 in pcDNA3.1-ZDHHC3 group were significantly increased,the expression of Bax was decreased,and the expres-sion of Bcl-2 was increased(P<0.01).Compared with model group and pcDNA3.1-GFP group,improved cell growth and decreased apoptotic rate were observed in pcDNA3.1-ZDHHC3 group(P<0.05).CONCLUSION:Palmitoyltransferase ZDHHC3 may play protective roles in CIRI by activating synapse-related proteins,regulating synaptic plasticity and al-leviating apoptosis.

关 键 词：脑缺血-再灌注损伤 棕榈酰转移酶ZDHHC3 突触可塑性 细胞凋亡 

分 类 号：R743[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]