Department of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, PR China.
Neurochem Int. 2013 Aug;63(2):69-79. doi: 10.1016/j.neuint.2013.04.011. Epub 2013 May 9.
Cerebral hypoxic preconditioning (HPC) provides neuroprotection by intracellular signaling pathways. We previously demonstrated that novel protein kinase Cε (nPKCε) activation participated in cerebral HPC development. In this study, we explore the role of nPKCε in HPC-induced neuroprotection against middle cerebral artery occlusion (MCAO)-induced ischemic injury and identify its possible signaling molecules. A total of 131 adult male BALB/c mice were divided into eight groups: normoxic control (n=9), HPC (n=9), HPC+εV1-2 (n=13), Sham (n=19), HPC+sham (n=6), Ischemia (I, 6h MCAO, n=31), HPC+I (n=25) and HPC+εV1-2+I (n=19). nPKCε specific inhibitor εV1-2 was administered via intracerebroventricular injection. Western blot, 2,3,5-triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were applied to determine nPKCε membrane translocation, infarction volume and programmed cell death (PCD), respectively. Two-dimensional gel electrophoresis (2-De) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify nPKCε-interacting proteins, followed by bioinformatics analysis of genee ontology (GO) to predict nPKCε-specific signaling pathways. Our results showed that HPC attenuates MCAO-induced brain injuries and stabilized nPKCεmembrane translocation in peri-infarct region, which was abolished by nPKCε-speecific inhibitor εV1-2. Proteomics analysis revealed 8 up- and 3 down-regulated nPKCε-interacting proteins both in cytosolic and particulate fractions of HPC mouse brain. GO analysis predicted 25 significant nPKCε-specific signaling pathways among the 16 identified nPKCε-interacting proteins in brain of HPC mice. This study is the first to report multiple nPKCε-interacting proteins and their signaling pathways in HPC mouse brain, suggesting that nPKCε signaling molecules is responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice.
脑缺氧预处理（HPC）通过细胞内信号通路提供神经保护。我们之前的研究表明，新型蛋白激酶 Cε（nPKCε）的激活参与了脑 HPC 的发展。在这项研究中，我们探讨了 nPKCε 在 HPC 诱导的神经保护中的作用，以对抗大脑中动脉闭塞（MCAO）引起的缺血性损伤，并确定其可能的信号分子。共有 131 只成年雄性 BALB/c 小鼠被分为八组：常氧对照组（n=9）、HPC 组（n=9）、HPC+εV1-2 组（n=13）、假手术组（n=19）、HPC+假手术组（n=6）、缺血组（I，6h MCAO，n=31）、HPC+I 组（n=25）和 HPC+εV1-2+I 组（n=19）。通过脑室内注射给予 nPKCε 特异性抑制剂 εV1-2。应用 Western blot、2,3,5-三苯基氯化四氮唑染色和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记法分别确定 nPKCε 膜转位、梗死体积和程序性细胞死亡（PCD）。二维凝胶电泳（2-De）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）用于鉴定 nPKCε 相互作用蛋白，然后通过基因本体（GO）的生物信息学分析预测 nPKCε 特异性信号通路。我们的结果表明，HPC 减轻了 MCAO 引起的脑损伤，并稳定了 HPC 小鼠梗死周边区 nPKCε 的膜转位，这一作用被 nPKCε 特异性抑制剂 εV1-2 所阻断。蛋白质组学分析显示，在 HPC 小鼠的脑细胞质和颗粒部分，nPKCε 相互作用蛋白中有 8 个上调和 3 个下调。GO 分析预测，在 HPC 小鼠脑中鉴定的 16 个 nPKCε 相互作用蛋白中，有 25 个是 nPKCε 特异性的信号通路。这项研究首次报道了 HPC 小鼠脑中的多个 nPKCε 相互作用蛋白及其信号通路，表明 nPKCε 信号分子负责 HPC 诱导的对小鼠脑缺血损伤的神经保护作用。
