脑衰蛋白反应调节蛋白-2参与低氧预适应减轻小鼠脑缺血损伤

目的 研究参与低氧预适应(HPC)的经典型蛋白激酶C II(cPKC II)相互作用的脑衰蛋白反应调节蛋白-2(CRMP-2) 对缺血脑是否有保护作用。方法 成年雄性BALB/c小鼠随机分为常氧(Nor)、HPC、常氧假手术(Nor+sham)、HPC假手术(HPC+sham)、常氧缺血(Nor+I)和HPC缺血(HPC+I)等6组(每组n=6)。应用小鼠整体HPC和脑中动脉梗死(MCAO)致脑局部缺血模型，结合免疫沉淀、双向凝胶电泳和质谱等技术，分离和鉴定与cPKC II相互作用蛋白；利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western blot)技术，分析CRMP-2磷酸化和蛋白降解水平在脑HPC和缺血中的变化。结果 与Nor组比，HPC鼠脑皮层组织内有10种与cPKC? II相互作用蛋白的表达发生了明显变化，其中CRMP-2在膜相关蛋白组分中的表达量升高，而在胞质蛋白组分中的表达量降低。在脑缺血模型中，与Nor+Sham组相比，Nor+I组小鼠脑皮层缺血核心区(Ic)CRMP-2磷酸化水平明显降低(p<0.05, n=6)；与Nor+I组相比，HPC+I组小鼠脑皮层Ic区内CRMP-2磷酸化水平明显增高(p<0.05, n=6)。脑缺血可导致CRMP-2发生水解并伴随着大量55-ku水解片段(BDP)的出现，但与Nor+I组相比，HPC+I组小鼠脑皮层缺血半影区(P)内CRMP-2水解片段减少，水解率明显降低(p<0.05, n=6)。结论 CRMP-2参与了 HPC缓解小鼠脑缺血Ic区内CRMP-2磷酸化水平的降低和减少P区内CRMP-2水解片段从而减轻缺血脑组织的损伤。

CRMP-2 is involved in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injuries of mice

Objective To investigate whether conventional protein kinase C (cPKC ) βⅡ-interacting collapsin response mediator protein-2 (CRMP-2) provides neuroprotection against cerebral ischemic (I) injuries. Methods Male BALB/c mice were randomly divided into normoxic control (Nor) , HPC, Nor + Sham, HPC + Sham, Nor + I and HPC + I groups (n = 6 per group). Using our HPC and MCAO mouse models, we applied immunoprecipita-tion, two-dimensional electrophoresis and mass spectrometry to characterize cPKCβⅡ-interacting proteins and combined with SDS-PAGE and Western blot to quantitatively analyze CRMP-2 phosphorylation and degradation levels in the brain of mice after HPC and MCAO. Results The expression level of 10 cPKCβⅡ-interacting proteins changed obviously in cerebral cortex of HPC mice when compared with Nor group. One of these proteins, CRMP-2 protein level increased in particulate fraction and decreased in cytosolic fraction of cerebral cortex of HPC mice. CRMP-2 phosphorylation level in ischemic core (Ic) of cerebral cortex decreased significantly ( P < 0. 05 , n = 6) as compared with that of Nor + sham group, but CRMP-2 phosphorylation level in HPC +I group increased significantly as compared with that of Nor +I group ( P < 0. 05, n = 6). In ischemic cortex, CRMP-2 degradation (proteolysis) was observed as the appearance of 55 ku breakdown products (BDP). However, the CRMP-2 degradation level, BDPs products decreased significantly in penumbra ( P) of ischemic cortex from HPC +I group when we compared with that of Nor +I group (P < 0. 05, n = 6 ). Conclusion CRMP-2 is involved in attenuating the decrease of CRMP-2 phosphorylation in ischemic core and in inhibiting its degradation in penumbra of cerebral cortex of mice thereby to lessen the ischemic injuries.