Chiang Ming-Chou, Ashraf Qazi M, Ara Jahan, Mishra Om P, Delivoria-Papadopoulos Maria
Department of Pediatrics, Chang Gung University College of Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Neurosci Lett. 2007 Jun 21;421(1):67-71. doi: 10.1016/j.neulet.2007.05.024. Epub 2007 May 24.
We have previously shown that the activity and the expression of caspase-9 and caspase-3 were increased during hypoxia in the cerebral cortex of newborn piglets. The present study was conducted to test the hypothesis that the hypoxia-induced activation of caspase-3 in the cerebral cortex of newborn piglets is mediated by caspase-9. Twenty-two newborn piglets were randomly assigned to four groups: normoxic (Nx), normoxic pretreated with a selective caspase-9 inhibitor, Z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethyl ketone (Z-LEHD-FMK) (Nx+LEHD), hypoxic (Hx), and hypoxic pretreated with Z-LEHD-FMK (Hx+LEHD). Cerebral tissue hypoxia was confirmed biochemically by measuring ATP and phosphocreatine. Caspase-9 and -3 activities were determined spectrofluorometrically. The expression of caspase-9 and -3 proteins was measured by Western blot analysis using active enzyme specific antibodies. Cytosolic caspase-9 activity (nmol/mg protein/h) was 3.70+/-0.40 in Nx, 3.56+/-0.31 in Nx+LEHD (p=NS versus Nx), 4.99+/-0.64 in Hx (p<0.05 versus Nx), and 3.73+/-0.80 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). Cytosolic caspase-3 activity (nmol/mg protein/h) was 7.80+/-1.17 in Nx, 8.15+/-0.87 in Nx+LEHD (p=NS versus Nx), 13.07+/-0.78 in Hx (p<0.05 versus Nx), and 10.05+/-2.09 in Hx+LEHD (p<0.05 versus Hx) The density (ODxmm(2)) of active caspase-9 protein was 18.52+/-1.89 in Nx, 20.53+/-1.12 in Nx+LEHD (p=NS versus Nx), 32.36+/-5.03 in Hx (p<0.05 versus Nx), and 19.94+/-3.59 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). The density (ODxmm(2)) of active caspase-3 protein was 55.87+/-8.73 in Nx, 55.69+/-8.18 in Nx+LEHD (p=NS versus Nx), 94.10+/-12.05 in Hx (p<0.05 versus Nx), and 56.12+/-14.56 in Hx+LEHD (p<0.05 versus Hx, p=NS versus Nx). These data show that administration of a selective caspase-9 inhibitor, Z-LEHD-FMK, prior to hypoxia prevents the hypoxia-induced increase in caspase-3 activity and the expression of active caspase-3 protein. We conclude that the hypoxia-induced activation of caspase-3 during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9.
我们之前已经表明，新生仔猪大脑皮层在缺氧期间，半胱天冬酶-9（caspase-9）和半胱天冬酶-3（caspase-3）的活性及表达会增加。本研究旨在验证以下假说：新生仔猪大脑皮层中缺氧诱导的半胱天冬酶-3激活是由半胱天冬酶-9介导的。22只新生仔猪被随机分为四组：常氧组（Nx）、用选择性半胱天冬酶-9抑制剂Z-亮氨酸-谷氨酸（甲酯）-组氨酸-天冬氨酸（甲酯）-氟甲基酮（Z-LEHD-FMK）预处理的常氧组（Nx+LEHD）、缺氧组（Hx）以及用Z-LEHD-FMK预处理的缺氧组（Hx+LEHD）。通过测量三磷酸腺苷（ATP）和磷酸肌酸，生化方法证实脑组织缺氧。用荧光分光光度法测定半胱天冬酶-9和-3的活性。使用活性酶特异性抗体，通过蛋白质印迹分析测量半胱天冬酶-9和-3蛋白的表达。常氧组（Nx）胞质半胱天冬酶-9活性（nmol/毫克蛋白/小时）为3.70±0.40，Nx+LEHD组为3.56±0.31（与Nx相比，p=无显著性差异），缺氧组（Hx）为4.99±0.64（与Nx相比，p<0.05），Hx+LEHD组为3.73±0.80（与Hx相比，p<0.05，与Nx相比，p=无显著性差异）。常氧组（Nx）胞质半胱天冬酶-3活性（nmol/毫克蛋白/小时）为7.80±1.17，Nx+LEHD组为8.15±0.87（与Nx相比，p=无显著性差异），缺氧组（Hx）为13.07±0.78（与Nx相比，p<0.05），Hx+LEHD组为10.05±2.09（与Hx相比，p<0.05）。活性半胱天冬酶-9蛋白的密度（OD×mm²）在常氧组（Nx）为18.52±1.89，Nx+LEHD组为20.53±1.12（与Nx相比，p=无显著性差异），缺氧组（Hx）为32.36±5.03（与Nx相比，p<0.05），Hx+LEHD组为19.94±3.59（与Hx相比，p<0.05，与Nx相比，p=无显著性差异）。活性半胱天冬酶-3蛋白的密度（OD×mm²）在常氧组（Nx）为55.87±8.73，Nx+LEHD组为55.69±8.18（与Nx相比，p=无显著性差异），缺氧组（Hx）为94.10±12.05（与Nx相比，p<0.05），Hx+LEHD组为56.12±14.56（与Hx相比，p<0.05，与Nx相比，p=无显著性差异）。这些数据表明，在缺氧前给予选择性半胱天冬酶-9抑制剂Z-LEHD-FMK可防止缺氧诱导的半胱天冬酶-3活性增加以及活性半胱天冬酶-3蛋白的表达。我们得出结论，新生仔猪大脑皮层在缺氧期间缺氧诱导的半胱天冬酶-3激活是由半胱天冬酶-9介导的。
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