亚低温降低新生大鼠缺氧缺血脑损伤半胱氨酸蛋白酶活性

Moderate hypothermia attenuates cysteinyl aspartate-specific proteinase activity after hypoxia-ischemic brain damage in neonatal rat

目的 探讨亚低温治疗对新生大鼠缺氧缺血脑损伤 (HIBD)脑组织天冬氨酸特异酶切的半胱氨酸蛋白酶 3(caspase 3)活性的影响及意义。 方法 将HIBD模型鼠随机分为常温缺氧缺血 (IN)组 (肛温 37℃ )和亚低温缺氧缺血 (IH)组 (肛温 33℃ ) ;对照组为假手术动物 ,分为常温对照 (CN)组和亚低温对照 (CH)组。采用显色底物天冬氨酸 谷氨酸 缬氨酸 天冬氨酸 7 氨基 4 三氟甲基香豆素 (DEVD AFC)测定caspase 3酶的活性 ,并采用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法 (TUNEL)检测脑细胞凋亡。结果 缺氧缺血侧大脑组织caspase 3活性逐渐增高 ,以IN组最明显 ,2 4h达高蜂 (34.7± 3.2 ) ,然后逐渐下降 ,但 4 8h(12 .9± 4 .1)和72h(4 .2± 0 .9)后仍有明显增高 ;亚低温可明显降低caspase 3活性的增高 ,以亚低温治疗 2 4h(2 .4± 0 .5 )最明显 ,与IN组比较 ,差异有显著性 (P <0 .0 0 1) ;72h亚低温还可显著降低脑细胞凋亡的发生率 ,为 (6 .4± 1.7) % ,与IN组的 (2 5 .3± 1.5 ) %相比 ,差异有显著性 (P <0 .0 1) ;72h亚低温对细胞坏死无明显改善 ,细胞坏死率为(11.2± 0 .7) % ,与IN组的 (13.0± 1.4 ) %相比 ,差异无显著性 (P >0 .0 5 )。caspase 3活性与脑细胞凋亡发生率呈正相关 (r =0 .78,P

Objective To investigate the effects of moderate hypothermia on cysteinyl aspartate-specific proteinase (caspase-3) activity and its significances after hypoxia-ischemic brain damage (HIBD) in neonatal rat. Methods Postnatal d 7 Sprague-Dawley neonatal rats were subjected to unilateral ligation of the left carotid artery followed by exposure to hypoxia in 8% O 2 for 2 h , and the rats were randomly divided into normothermia group(rectal temperature 36℃) and moderate hypothermia group(rectal temperature 32℃). Sham-operated rats in the same litter were control groups including normothermic control group and moderate hypothermia control group. 0, 2, 6, 24, 48, 72 h after hypoxia-ischemic(HI) insult, the ipsilateral cortex and hippocampus were dissected and homogenized for caspase-3 assay. Caspase-3 activity was measured by specific cleavage of the fluorescent DEVD substrate peptide conjugated to 7-amino-4-trifluoromethyl coumarin (AFC), and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining (TUNEL) method was used to detect neuronal apoptosis. Results Caspase-3 activity was gradually increased from 2 h (2.2±1.1) after HI insult and reached peak at 24 h (34.7±3.2 ) in the normothermic group and then slowly decreased, but there were still moderate increase at 48 and 72 h (12.9±4.1,4.2±0.9,respectively )after HI in the ipsilateral cortex and hippocampus. In contrast to the normothermic group, moderate hypothermia treatment resulted in a significant reduction in caspase-3 activity in the cortex and hippocampus ipsilateral to the carotid ligation. The optimal effect was produced at 24 h after initiation of hypothermia, and there was a significant difference between these two groups(2.4±0.5 vs 34.7±3.2,F=32.1, P<0.001). A time-dependent increase in the number of TUNEL-positive neuron in the hemisphere ipsilateral to carotid ligation was noted in normothermic group. Comparing with normothermia, moderate hypothermia can substantially decrease the TUNEL-positive neuron ratio after HI insult at 72 h[(6.4 ±1.7)% vs (25.3±1.5)%,F=15.6,P<0.01], but there was no significant amelioration of necrosis of neuron ratio[(11.2±0.7)% vs (13.0±1.4)%,P>0.05], and a close correlation between caspase-3 activity and TUNEL-positive neuron ratio was found. Conclusions Taken together, these results suggest that the mechanism for the in vivo attenuation effects of neuronal apoptosis by moderate hypothermia is through inhibition of caspase-3 activation and subsequent steps leading to neuronal apoptosis.