钙蛋白酶通过降解突触蛋白Ⅰ介导脑出血后继发性神经元损伤

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（50 μmol/L）和MDL28170
（10 μmol/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。

Calpain Induces Secondary Neuronal Injury after Intracerebral Hemorrhage via Degradation of Synapsin I

Objective To investigate the role of calpain in secondary neuronal injury after intracerebral hemorrhage.Methods Experimental intracerebral hemorrhage（ICH） was induced in mice by intraparenchymal injection of autologous blood. The animals were randomly separated into 4 groups：0 h group（control group）, 2 h group, 4 h group, and 24 h group. Each group contains 10 mice. The activity of calpain in the brain tissues were evaluated at different time points. The degradation of synapsin Ⅰ was analyzed by Western blot. The cleavage of recombinant synapsin Ⅰ was analyzed after incubation with active calpain. The effects of exogenous calpain and calpain inhibitors ALLN and MDL28170 on PC12 cells were analyzed by MTT assay. The expression of synapsin Ⅰ was determined by Western blot.Results The activity of calpain increased in a time-dependent manner, with peak activation at 24 h. 0 h, 2 h, 12 h, and 24 h after surgery, the calpain activity reading was 234.32, 343.87, 425.29, and 597.36, respectively. The calpain activity of 12 h group and 24 h group was signifi cantly higherthan the 0 h group（P〈0.05）. The expression of synapse-associated protein synapsin Ⅰ decreased as the activity of calpain increased, indicating synapsin Ⅰ might be cleaved by calpain. The protein level of synapsin Ⅰ decreased to 67% of the control group（P〈0.01）. To verify the cleavage of synapsin Ⅰ by calpain, purifi ed recombinant synapsin Ⅰ was incubated with active calpain in vitro. Calpain induced the degradation of synapsin Ⅰ. Synapsin Ⅰ level decreased to 57.75%（P =0.001） of the control group after 30 min incubation. The in vitro cleavage of synapsin Ⅰ by calpain was inhibited by calpain inhibitors. The level of synuclein Ⅰ was 87.00% and 84.75% in the present of ALLN and MDL28170, respectively（P〈0.05 compared to the calpain group）. Exogenous calpain induced cell toxicity in neuronal differentiated PC12 cells. The cell viability decreased to 58.25% of the control group after 12 h incubation, In