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。
Objective To validate the role of conventional protein kinase C (cPKC)γ in hypoxic precondition (HPC)- induced neuroprotection against brain ischemie injury, and determine cPKC5, -specific signaling proteins in penumbra of mice following ischemic stroke by using ePKCγ inhibitor and HPC mouse model. Methods Healthy adult male BALB/c mice were randomly divided into five groups, including normoxic sham, normoxie 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, Trc staining, cPKCγ immunoprecipitation, two-dimensional eleetrophoresis (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 contralaleral cortex of HPC + (306983 +I mice (P 〈 0. 05, n = 6 per group), cPKCγ inhibitor Go6983 abolished HPC-induced reduttion in infarction size of mice with ischenlic stroke ( P 〈 0. 05, n = 6 per group). In addition, Go6983 could inhibit the intcraction between cPKCγ and partial interacting proteins in ischemic pcnumbra of HPC + I mice, including superoxide dismutase [ Cu-Zn ] , DJ-I , UMP- CMP kinase, adenyla/e kinase isoenzyme 1, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) , interleukin-16 and ]cleat shock protein 70 (HSP70). Conclusion cPKCγ participates HPC-induced neuroprotcction though regulating its interaction with partial interacting proteins in ischemie penumbra of mice following ischemie stroke.
出处
《首都医科大学学报》
CAS
2013年第5期698-703,共6页
Journal of Capital Medical University
基金
国家自然科学基金项目(31071048
31171147
31200895)
科技部973计划前期研究专项(2011CB512109)
北京市自然科学基金项目(7132025)~~