半胱氨酸天冬氨酸蛋白酶3对缺血再灌注大鼠脑海马神经元凋亡的影响

背景半胱氨酸天冬氨酸蛋白酶3在正常细胞中以酶原形式存在,受凋亡刺激因素作用后能够被激活从而引起细胞凋亡.目的通过检测脑海马组织胞质S-100中半胱氨酸天冬氨酸蛋白酶3的活性及海马区神经元凋亡情况,探讨全脑缺血再灌注后脑海马神经元凋亡与半胱氨酸天冬氨酸蛋白酶3的关系.设计随机对照实验.单位解放军第三军医大学西南医院急救部.材料实验于1999-01/04在解放军第三军医大学西南医院完成.选取雄性Wistar大鼠182只,随机分为3组假手术组14只,脑缺血再灌注组84只,乙酰天冬氨酰谷氨酰缬氨酰天冬氨酸乙醛(acetyl-asp-glu-valasp-aldehyde,AC-DEVD-CHO)治疗组84只,后两组均设立缺血再灌注8,24,48,72,120,168 h 6个时相点,每个时相点14只大鼠.方法脑缺血再灌注组、AC-DEVD-CHO治疗组建立大鼠全脑缺血20 min再灌注模型,分别于再灌注后8,24,48,72,120,168 h处死取海马组织;假手术组施行麻醉及手术,但不夹闭颈总动脉和烧灼椎动脉,术后观察72 h后处死取海马组织备检.以单位质量标本于单位时间内裂解产生的氨甲基香豆素量表示标本中半胱氨酸天冬氨酸蛋白酶3的活性.各组不同时相点脑组织切片封固后在荧光显微镜下于330～350 nm处观察脑海马细胞凋亡情况.主要观察指标①各组脑缺血再灌注后不同时相点海马组织S-100中半胱氨酸天冬氨酸蛋白酶3活性变化.②各组脑缺血再灌注后不同时相点海马细胞凋亡的情况.③半胱氨酸天冬氨酸蛋白酶3活性与海马区神经元凋亡的关系.结果实验纳入182只大鼠,脱落14只,共168只大鼠进入结果分析.①各组脑缺血再灌注后不同时相点海马组织S-100中半胱氨酸天冬氨酸蛋白酶3活性变化假手术组术后观察72时为0.与脑缺血再灌注组比较,AC-DEVD-CHO治疗组于再灌注24,48,72,120,168 h均明显降低[(1.71±0.03),(1.22±0.03);(2.77±0.09),(1.59±0.7);(5.54±0.51),(2.3±0.19);(6.28±1.71),(3.43±0.46);(3.11±1.21),(1.73±0.14)nkat/kg;P＜0.05或0.01].②各组脑缺血再灌注后不同时相点海马细胞凋亡的情况每400倍视野下,假手术组术后观察72 h为(1.2±0.4)个.与脑缺血再灌注组比较,AC-DEVD-CHO治疗组于再灌注24,48,72,120,168 h均明显降低[(6.4±1.7),(2.8±0.8);(11.8±1.3),(5.8±1.9);(19.8±3.1),(10.0±1.9);(31.2±5.9),(16.4±2.4);(19.8±2.3),(9.0±2.3)个/400倍视野;P＜均0.01].③半胱氨酸天冬氨酸蛋白酶3活性与海马区神经元凋亡的关系脑缺血再灌注组、AC-DEVD-CHO治疗组均行直线相关分析,二者的变化呈显著正相关(r分别为0.935 6及0.980 0,P均＜0.01).结论半胱氨酸天冬氨酸蛋白酶3的激活是引起海马神经元调亡的主要因素之一,在缺血再灌注大鼠脑海马神经元凋亡中起着重要作用.

Influence of caspase-3 on hippocampal neuronal apoptosis following ischemia-reperfusional injury in rats

BACKGROUND: Caspase-3 exists in normal cell in form of zymogen and is capable of stimulating cell apoptosis after activated by apoptosis inducing factors.OBJECTIVE: To observe the activity of caspase-3 in hippocampal cytosolic S-100 and neuronal apoptosis in hippocampal regions, so as to discuss the relationship between hippocampal neuronal apoptosis and caspase3 activity during the whole brain ishcemic-reperfuasional injury.DESIGN: Randomized controlled experiment.SETTING: Emergency Department of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA.MATERIALS: This experiment was carried out at Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA from January to April 1999. Totally 182 male Wistar rats were randomly divided into 3 groups: namely sham operation group of 14 rats, cerebral IR group of 84, rats acetyl-asp-glu-val-asp-aldehyde (AC-DEVD-CHO) treatment group of 84 rats, rats in the latter two groups were then subdivided into IR 8, 24, 48, 72, 120 and 168 hours time points subgroups with 14 rats in each.METHODS: The whole brain ischemia 20 minutes and reperfusional model was established on rats in brain IR group and Ac-DEVD-CHO treatment group, and rats were executed separately at post-reperfusional 8, 24,48, 72, 120 and 168 hours for obtaining hippocampal specimen; rats in sham operation group were only underwent anesthesia and operation without common carotid arterial occlusion and burns of vertebral artery, they were executed at 72 hours after operation and hippocampal specimen was obtained. The quantity of amino-methylcoumarin that was produced from the same mass of specimen within same decomposition time was used to reflect the activity of caspase-3. Brain slices that were obtained from different time points were stained and embedded for observing the hippocampal cell apoptosis under fluorescence microscope at 330-350 nm.MAIN OUTCOME MEASURS: ① The caspase-3 activity in hippocampal S-100 in different post-IR time point groups. ② The hippocampal cell apoptosis in different post-IR time point groups. ③ relationship between caspase-3 activity and neuronal apoptosis in hippocampal regions.RESULTS: Totally 182 rats were enrolled in this experiment, 14 rats got lost, thereby date of 168 rats was entered the result analysis. ① The changes of caspase-3 activity in hippocampal S-100 in different post-IR time point groups: There was no change in sham operation group at postoperative 72 hours. In contrast with cerebral IR group, there were obvious reduction in Ac-DEVD-CHO treatment group at post-reperfusional 24, 48,72, 120 and 168 hours [(1.71±0.03, 1.22±0.03; 2.77±0.09, 1.59±0.7;5.54±0.51, 2.3±0.19, 6.28±1.71, 3.43±0.46; 3.11±1.21, 1.73±0.14) nkat/kg;P ＜ 0.05 or 0.01]. ② The hippocampal cell apoptosis in different post-IR time point groups: Under 400× field of vision, the number of apoptotic cells in sham operation group was 1.2±0.4 cells at postoperative 72 hours.It was lower in Ac-DEVD-CHO treatment group at post-reperfusional 24,48, 72, 120 and 168 hours than cerebral IR group [(6.4±1.7, 2.8±0.8;11.8±1.3, 5.8±1.9; 19.8±3.1, 10.0±1.9; 31.2±5.9, 16.4±2.4; 19.8±2.3, 9.0±2.3)cells/400× field of vision; P ＜ 0.01]. ③ Relationship between caspase-3activity and neuronal apoptosis in hippocampal regions: It was proved of linear correlation in cerebral IR group and Ac-DEVD-CHO treatment group,displaying significantly positive correlation r= 0.935 6 or 0.980 0, P ＜ 0.01).CONCLUSION: Caspase-3 activation is one of the major inducer for hippocampal neuronal apoptosis, playing important role in hippocampus neuronal apoptosis in rats during IR injury.