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目的观察钙蛋白酶在实验性脑出血后继发性神经元损伤中的作用及其机制。方法脑内注射自体血液法制作小鼠脑出血模型,将10周龄雄性C57BL/6J小鼠实验动物随机分为4组:0 h组(对照组)、2 h组、12 h组和24 h组,每组10只小鼠。收集血肿周围脑组织后检测血肿周围组织钙蛋白酶活性水平,免疫印迹法检测突触蛋白Ⅰ表达水平。重组的突触蛋白Ⅰ与钙蛋白酶共孵育后检测其降解过程,并观察钙蛋白酶抑制剂ALLN和MDL28170的作用。在体外培养的神经元样大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)中加入钙蛋白酶,观察其细胞损伤作用和对突触蛋白Ⅰ的降解。结果脑出血后2 h血肿周围脑组织钙蛋白酶活性升高,至24 h达到高峰。脑出血0 h、2 h、12 h和24 h后钙蛋白酶活性读数分别为234.32、343.87、425.29和597.36,12 h组和24 h组与对照组相比差异具有显著性(P<0.05)。同时突触蛋白Ⅰ水平逐渐降低,提示其降解。至血肿形成后24 h下降到对照组的67.00%(P<0.01)。纯化的重组突触蛋白Ⅰ与钙蛋白酶共孵育后出现降解,孵育30 min后突触蛋白Ⅰ含量下降至对照组(0 h组)的57.75%(P=0.001),在钙蛋白酶抑制剂ALLN(50μmol/L)和MDL28170(10μmol/L)存在的情况下,突触蛋白Ⅰ水平分别为对照组的87.00%和84.75%(与单纯钙蛋白酶处理组30 min组相比,P<0.05)。外源性钙蛋白酶与PC12细胞共孵育12 h后细胞活力下降到对照组的58.25%(P<0.01),而ALLN和MDL28170处理组细胞活力升高到对照组的83.00%和80.25%(与单纯钙蛋白酶处理组相比,P<0.01)。结论钙蛋白酶通过降解突触蛋白Ⅰ参与脑出血后的继发性神经损伤,有可能成为脑出血的治疗靶点。
Objective To investigate the role of calpain in secondary neuronal injury after experimental intracerebral hemorrhage and its mechanism. Methods The model of intracerebral hemorrhage was induced by intracerebral injection of autologous blood. The 10-week-old male C57BL / 6J mice were randomly divided into 4 groups: 0 h group (control group), 2 h group, 12 h group and 24 h Groups of 10 mice per group. The brain tissue around the hematoma was collected to detect the level of calpain activity around the hematoma, and the expression of synaptophysin Ⅰ was detected by Western blotting. The recombinant synaptophysin Ⅰ was incubated with calpain to detect the degradation process, and the effects of calpain inhibitor ALLN and MDL28170 were observed. Calpain was added to adrenal pheochromocytoma cells (PC12 cells) cultured in vitro to observe the cell injury and the degradation of synapsin Ⅰ. Results After 2h of intracerebral hemorrhage, the activity of calpain increased around the hematoma and peaked at 24 h. Calpain activity at 0 h, 2 h, 12 h and 24 h after intracerebral hemorrhage was 234.32,343.87,425.29 and 597.36, respectively. There was significant difference in calpain activity between the two groups at 24 h and 24 h (P <0.05). At the same time, the level of synaptophysin Ⅰ gradually decreased, suggesting its degradation. 24 h after hematoma formation to 67.00% of the control group (P <0.01). The purified recombinant synaptophysin Ⅰ was degraded after co-incubation with calpain and the content of synaptophysin Ⅰ decreased to 57.75% (P = 0.001) in the control group (0 h group) after 30 min of incubation. In the calpain inhibitor ALLN ( The levels of synaptophysin Ⅰ were 87.00% and 84.75% in the control group (P <0.05 compared with the 30 min group in the calpain alone group) in the presence of 50μmol / L MDL28170 and 10μmol / L respectively. Exogenous calpain incubated with PC12 cells for 12 h decreased cell viability to 58.25% of the control group (P <0.01), while the cell viability of ALLN and MDL28170 increased to 83.00% and 80.25% of the control group Calpain treatment group, P <0.01). Conclusions Calpain may be a therapeutic target for cerebral hemorrhage by degrading synaptic protein I in secondary neuronal injury after intracerebral hemorrhage.