大鼠脑缺血再灌注后溶酶体组织蛋白酶L与细胞凋亡的关系

The relationship between cathepsin L and apoptosis cell in rats after cerebral ischemia reperfusion

目的探讨大鼠脑缺血再灌注后溶酶体组织蛋白酶L（CathepsinL）与细胞凋亡的关系。方法选取10～12周龄的清洁级健康雄性Sprague—Dawley（SD）大鼠60只，体重（280±20）g，依据随机数字表法将大鼠随机分为假手术组（Sham组）10只、脑缺血再灌注组（模型组）25只、Cathep-sinL抑制剂Z．FY．DMK干预组（CLI组）25只，模型组、CLI组大鼠分别按6、12、24及48h每个时间点每组5只大鼠随机分为四个亚组。采用改良longa线栓法制作大鼠大脑中动脉缺血再灌注损伤模型（MCAO），CLI组于术前30rain侧脑室穿刺注入Z—FY—DMK（20μg／1μl×5μ1），Sham组及模型组于术前30min侧脑室注入浓度为10ml／L的DMSO5仙l。对大鼠运用TUNEL法检测缺血侧大脑皮质神经细胞凋亡变化，Westernblotting法检测半暗带区相应时间点CathepsinL、Caspase-3蛋白表达变化。结果在脑缺血侧皮质区的凋亡细胞检测中，假手术组脑组织中凋亡细胞几乎很少见，模型组6h可见凋亡神经细胞，12、24、48h逐渐增多，呈上升趋势；CLI组在6、12、24、48h观察到凋亡细胞明显少于对应时间点的模型组（P〈0．05）。在脑缺血侧皮质区的Westernblotting检测相关蛋白中，假手术组的脑组织中可以检测到少量的CathepsinL蛋白表达；模型组CathepsinL蛋白均在再灌注6h开始上升，12、24h达到高峰，48h仍保存高水平；与模型组比较，CLI组CathepsinL蛋白表达在各时间点均明显减弱（P〈0．05）。结论CathepsinL可能参与了大鼠短暂性前脑缺血再灌注损伤后caspase、3细胞凋亡通路。

Objective To investigate the relationship between cathepsin L and apoptosis cell in rats after cerebral ischemia reperfusion. Methods Sixty healthy male Sprague-Dawley Rats （ 10 - 12 weeks old, 260 - 300 g） were chosen. Based on the random number table method, the rats were randomly divided into sham-operated control group （ Sham group, n = 10）, ischemia-reperfusion group （ model group, n = 25）, and Z-FY-DMK intervention group （CLI group, n =25）. Rats were randomly divided into 6 h, 12 h, 24 h, and 48 h four subgroups in model group and CLI group, respectively. Modified transient middle cere- bral artery occlusion was made as Longa described, the intervention groups were injected intracerebroven- tricularly Z-FY-DMK （20 μg / lμl x5 μl） preoperative 30 min prior to surgery, Sham group and schemia reperfusion injury （IRI） group were injected intracerebroventricularly dimethyl sulfoxide （DMSO） 5 μl （10 ml/L） at the same time. Cell apoptosis was detected by terminal dexynucleotidyl transferase （TdT）-media- ted dUTP nick end labeling （TUNEL） straining. Western blotting was used to detect the expression of ca- thepsin L and caspase-3. Results In the cortical area of ischemic brain, apoptosis cells of sham operation group were rare, while apoptosis of nerve cells of model group with 6 hours reperfusion were visible, and were gradually increased in the order of 12 hours, 24 hours and 48 hours. At the same time point, the apop- tosis cells of CL intervention group （6 h, 12 h, 24 h, 48 h） were obviously less than model group （P 〈 0.05）. Western blotting found little visible cathepsin L protein expression in ischemic cerebral cortex pre- optic in the sham group. For model group, the cathepsin L expression initially increased in sub groups with 6 hours reperfusion, reached to a peak in sub groups with 12 hours and 24 hours, and remained a high level in sub groups with 48 hours reperfusion. Compared to model group, the cathepsin L expressions of CL inter-vention group were obviously decreased at all time points （ P 〈 O. 05 ）. Conclusions Cathepsin L may be involved in neuronal apoptosis by means of caspases 3 pathway.