半胱氨酸蛋白酶-3抑制剂对大鼠缺血再灌流脑区神经元凋亡的影响  

Effect of caspase-3 inhibitor in neuronal apoptosis in rat cerebral cortex after ischemia-reperfusion injury

作　　者：于士柱[1] 严莉[1] 王虔[1] 安同岭[1] 管欣琴[1] 

机构地区：[1]天津医科大学总医院

出　　处：《中华病理学杂志》2006年第3期165-170,共6页Chinese Journal of Pathology

基　　金：天津市科委重点攻关课题基金资助项目(953105711)

摘　　要：目的探讨半胱氨酸蛋白酶3(caspase3)抑制剂z DEVD fmk对大脑皮层缺血再灌流区神经元凋亡的影响。方法制备大脑中动脉栓塞再灌流大鼠模型,于再灌流前向治疗组缺血侧脑室注射z DEVD fmk(7μg/kg)。采用Western印迹分析、TUNEL和免疫组织化学染色(SPAB法)等方法,检测各组颞顶叶皮层缺血再灌流区caspase3表达和活化、多(ADP核糖)聚合酶(PARP)表达和切割灭活及神经元凋亡。结果未治疗组(A组)、二甲基亚砜对照组(B组)、z DEVD fmk治疗组(C组),再灌流1h及24h缺血脑区的caspase3前体含量分别为16.7±3.0、11.5±3.0、47.5±3.5及76.1±3.5、71.3±6.4、88.2±5.5;12000caspase3切割片段含量分别为8.2±2.3、9.4±1.2、4.3±1.6及59.0±6.3、60.5±7.2、17.3±2.8;PARP含量分别为12.6±3.0、13.9±2.0、53.7±4.1及67.5±8.6、61.1±6.6、93.6±4.1;24000PARP切割片段含量分别为6.0±0.7、6.6±1.2、3.6±1.1及27.4±2.6、25.8±3.2、12.1±2.8(相对灰度值);凋亡神经元密度分别为83.3±7.5、84.3±5.7、45.7±4.0及197.4±11.8、185.2±11.2、99.1±5.8(个/0.1mm2,x±s)。3组各自再灌流不同时间点缺血脑区以上5种指标的差异均有统计学意义(P<0.05～0.001);C组再灌流各时间点缺血脑区以上5种指标与A组及B组对应时间点比较,差异也均有统计学意义(P<0.05～0.001),但A、B两组间比较差异无统计学意义(P>0.05);各组再灌流不同时间点这5种指标的变化彼此间均呈正相关(r=0.630～0.942,P<0.01)。各组缺血再灌流脑区表达PARP的细胞主要是神经元,但3组间比较其密度差别不大。结论再灌流激发的caspase3表达和活化异常增加使PARP过度切割灭活,是再灌流导致缺血脑区受损神经元凋亡的重要分子机制;z DEVD fmk可通过抑制caspase3活性和自活化,减少PARP切割灭活,阻止受损神经元凋亡。Objective To investigate the effect of z-DEVD-fmk, a caspase-3 inhibitor on the neuronal apoptosis in ischemia-reperfusion region （IRR） of rat cerebral cortex. Methods Rats prepared by middle cerebral artery occlusion and reperfusion were used as the research model. The animals were divided into A group （untreated）, B group （DMSO control） and C group （treated with z-DEVD-fmk）. Before reperfusion, z-DEVD-fmk （7 μg/kg） was injected into the ischemic side of ventriculus cerebri of C group rats. The expression and activation of caspase-3, expression and cleavage of poly （ADP-ribose） polymerase （PARP）, and apoptotic neurons in the temporal-parietal cortex IRRs （SPAB method） of all the rats were studied using Western blotting, in situ apoptotic detection （TUNEL method） and immunohistochemistry. Results In the cerebral IRRs of A, B, C groups reperfused for 1 h and 24 h, the quantities of caspase-3 precursor were 16.7±3.0, 11.5 ±3.0 and 47.5 ±3.5, and 76. 1 ±3.5, 71.3 ±6.4 and 88.2 ±5.5, respectively ; the caspase-3 fragments （ 12 000） 8.2 ± 2. 3, 9. 4 ± 1.2 and 4.3 ± 1.6, and 59.0 ± 6. 3, 60. 5 ±7.2 and 17.3 ±2. 8, respectively; the PARP 12. 6 ±3.0, 13.9±2.0 and 53.7±4. 1, and 67.5 ± 8.6, 61.1 ±6. 6 and 93.6 ±4. 1, respectively; the PARP fragments （24 000） 6.0 ±0. 7, 6. 6 ±1.2, 3.6 ± 1.1, and 27.4 ± 2. 6, 25.8 ± 3.2, 12. 1 ±2. 8 （ relative quantity, x^- ± s ） ; the densities of apoptotic neurons83.3 ±7.5, 84.3 ±5.7 and45.7 ±4.0, and 197.4 ±11.8, 185.2±11.2 and99.1±5.8 （cell number/0. 1 mm^2, x^-± s ）. These results showed that in the cerebral IRRs of both A and B groups, all caspase-3 expression and activation, PARP expression and cleavage, and neuronal apoptosis were increased relevantly along with prolongation of the reperfusion time （ P 〈 0. 05 - 0. 001 ）. At each time point of the reperfusion, caspase-3 activation, PARP cleavage and neuronal apoptosis in the cerebral IRR of C group were significantly less than those of the for

关 键 词：脑缺血 再灌注损伤 半胱氨酸蛋白酶抑制剂 脱噬作用 

分 类 号：R743[医药卫生—神经病学与精神病学]