蛋白激酶Cγ在低氧预适应脑保护中的作用及其缺血性卒中鼠脑半影区内相互作用蛋白鉴定

doi:10.3969/j.issn.1006-7795.2013.05.013

首都医科大学学报 ›› 2013, Vol. 34 ›› Issue (5): 698-703.doi: 10.3969/j.issn.1006-7795.2013.05.013

蛋白激酶Cγ在低氧预适应脑保护中的作用及其缺血性卒中鼠脑半影区内相互作用蛋白鉴定

韩松, 张楠, 尹艳玲, 赵丽, 罗艳琳, 李俊发

首都医科大学基础医学院神经生物学系, 北京脑重大疾病研究院, 北京 100069

收稿日期:2013-07-16 出版日期:2013-10-21 发布日期:2013-10-22
通讯作者: 李俊发 E-mail:junfali@ccmu.edu.cn
基金资助:
国家自然科学基金项目(81273071,30901196);北京市优秀人才资助资助(2011D005018000011)。

Protein kinase Cγ play roles in hypoxic preconditioning-induced neuroprotection and determination of its interacting proteins in penumbra of mice with ischemic stroke

HAN Song, ZHANG Nan, YIN Yanling, ZHAO Li, LUO Yanlin, LI Junfa

Department of Neurobiology, School of Basic medical science, Beijing Institute for Brain Disorders, Capital Medical University, Beijing 1000069, China

Received:2013-07-16 Online:2013-10-21 Published:2013-10-22
Supported by:
This study was supported by National Natural Science Foundation of China (81273071,30901196);Excellent Person Project of Beijing (2011D005018000011).

摘要/Abstract

摘要：

目的验证经典型蛋白激酶C(conventional protein kinase C, cPKC)γ在低氧预适应(hypoxic preconditioning, HPC)诱导缺血脑保护中作用,同时利用HPC和cPKCγ阻断剂,鉴定脑缺血半影区内参与cPKCγ脑保护的信号蛋白。方法将健康成年雄性BALB/c小鼠,按数字表法随机分为常氧假手术(Sham)、常氧缺血(Ischemia, I)、二甲基亚砜溶剂处理后缺血(DMSO+I)、HPC预处理后缺血(HPC+I),以及cPKCγ抑制剂和HPC预处理后缺血(HPC+Go6983+I)等5组。应用小鼠整体HPC和脑中动脉阻塞(middle cerebral artery occlusion, MCAO)致缺血性脑卒中模型,借助蛋白印迹检测cPKCγ膜转位(激活)水平,2,3,5-氯化三苯基四氮唑(2,3,5-triph-enyltetrazolium chloride, TTC)染色观察脑梗死体积。采用cPKCγ免疫共沉淀、双向凝胶电泳和质谱等技术鉴定脑缺血半影区内cPKCγ相互作用蛋白结果。结果与常氧缺血I组相比,HPC+I组缺血核心区和半影区内cPKCγ膜转位水平明显增高,而cPKCγ抑制剂Go6983预处理则明显降低HPC+Go6983+I组缺血核心、半影区和对侧脑皮层内cPKCγ的膜转位水平(P<0.05, 每组n=6)。cPKCγ抑制剂Go6983明显减弱HPC降低缺血性卒中小鼠脑梗死体积的保护作用(P<0.05, 每组n=6)同时。Go6983明显抑制HPC+I组小鼠脑缺血半影区内cPKCγ与部分蛋白的相互作用,包括铜/锌超氧化物歧化酶(Cu/Zn superoxide dismutase, SOD)、DJ-1、UMP-CMP激酶、腺苷酸激酶、泛素末端水解酶(ubiquitin carboxyl-terminal hydrolase isozyme L1, UCHL1)、白介素-16 (interleukin-16, IL-16)和热休克蛋白70(heat shock protein 70, HSP70)。结论cPKCγ通过对缺血半影区内部分相互作用蛋白的调节,参与HPC对缺血性卒中小鼠脑的神经保护作用。

关键词: 低氧预适应, 缺血性脑卒中, 蛋白激酶C, 神经保护作用, 蛋白组学

Abstract:

Objective To validate the role of conventional protein kinase C (cPKC)γ in hypoxic precondition (HPC)- induced neuroprotection against brain ischemic injury, and determine cPKCγ -specific signaling proteins in penumbra of mice following ischemic stroke by using cPKCγ inhibitor and HPC mouse model.Methods Healthy adult male BALB/c mice were randomly divided into five groups, including normoxic sham, normoxic ischemia (I), ischemia following DMSO treatment (DMSO+I), ischemia post HPC (HPC+I), and ischemia after cPKCγ inhibitor Go6983 and HPC pretreatments. Using HPC and middle cerebral artery occlusion (MCAO)-induced ischemic stroke mouse models, the cPKCγ membrane translocation (activation) levels, cerebral infraction volum and cPKCγ -interacting proteins in ischemic penumbra were determined by Western blot, TTC staining, cPKCγ immunoprecipitation, two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), respectively. Results Compared with MCAO-induced ischemia (I) group, cPKCγ membrane translocation (activation) levels was increased significantly in ischemic core and penumbra of HPC+I group, but cPKCγ inhibitor Go6983 pretreatment could inhibit the increases of cPKCγ membrane translocation in ischemic core, penumbra and contralateral cortex of HPC+Go6983+I mice (P<0.05, n=6 per group). cPKCγ inhibitor Go6983 abolished HPC-induced reduction in infarction size of mice with ischemic stroke (P<0.05, n=6 per group). In addition, Go6983 could inhibit the interaction between cPKCγ and partial interacting proteins in ischemic penumbra of HPC+I mice, including superoxide dismutase [Cu-Zn], DJ-1, UMP-CMP kinase, adenylate kinase isoenzyme 1, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), interleukin-16 and heat shock protein 70 (HSP70). Conclusion cPKCγ participates HPC-induced neuroprotection though regulating its interaction with partial interacting proteins in ischemic penumbra of mice following ischemic stroke.

Key words: hypoxic preconditioning, ischemic stroke, protein kinase C, neuroprotection, proteomics

中图分类号:

R339.5

韩松, 张楠, 尹艳玲, 赵丽, 罗艳琳, 李俊发. 蛋白激酶Cγ在低氧预适应脑保护中的作用及其缺血性卒中鼠脑半影区内相互作用蛋白鉴定[J]. 首都医科大学学报, 2013, 34(5): 698-703.

HAN Song, ZHANG Nan, YIN Yanling, ZHAO Li, LUO Yanlin, LI Junfa. Protein kinase Cγ play roles in hypoxic preconditioning-induced neuroprotection and determination of its interacting proteins in penumbra of mice with ischemic stroke[J]. Journal of Capital Medical University, 2013, 34(5): 698-703.

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蛋白激酶Cγ在低氧预适应脑保护中的作用及其缺血性卒中鼠脑半影区内相互作用蛋白鉴定

Protein kinase Cγ play roles in hypoxic preconditioning-induced neuroprotection and determination of its interacting proteins in penumbra of mice with ischemic stroke

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