The role of Bcl-2 and Bax in hypoxia-induced caspase-3 activation in the fetal superior colliculus: in vivo optical

众所周知，胎儿大脑在分娩过程中会从严重缺氧中存活下来，尽管其生理机制尚不清楚。我们研究了Bcl-2 （caspase-3抑制因子）和Bax （caspase-3促进因子）在缺氧诱导的上丘（SC） caspase-3（最终触发凋亡因子）激活中的作用，胚胎第22天，胚胎上丘仍通过脐带与麻醉的屏障（聚氨酯，1.2-1.4 g/kg, i.p）相连。利用光学成像系统用荧光Caspase-3底物测定Caspase-3活性。阻断脐带诱导缺氧5分钟。初次闭塞再灌注脐血后，于第一次闭塞后2 ~ 5小时重复第2 ~ 5次闭塞再灌注。将Bcl-2-siRNA或Bax-siRNA电注入SC细胞。采用抗ssdna抗体检测胎儿SC细胞凋亡情况。脐带再灌注1 ~ 5 h后，细胞凋亡出现。Caspase-3活性在闭塞期间短暂升高，再灌注后持续时间较长。低剂量Bcl-2抑制剂对闭塞期和再灌注后caspase-3活性无影响。而高剂量Bcl-2抑制剂却意外抑制了caspase-3活性。Bax抑制剂对caspase-3活性无影响。此外，利用Bcl-2siRNA和Bax- sirna抑制Bcl-2和Bax合成，检测胎鼠caspase-3活性的变化。Bcl-2-siRNA在第2次和第3次再灌注后引起caspase-3活性的增加，统计学意义不显著，而Bax-siRNA引起caspase-3活性的显著降低。结果提示Bcl-2和Bax可能参与严重缺氧时未成熟脑细胞凋亡级联反应。

The fetal brain is known to be survival from severe hypoxia during delivery, although its physiological mechanism remains unclear. We studied the roles of Bcl-2 (caspase-3 suppressor) and Bax (caspase-3 accelerator) in the hypoxia-induced activation of caspase-3 (final triggering apoptosis factor) in the superior colliculus (SC) in a fetal rat (embryonic day 22), which was still connected with the anesthetized dam (urethane, 1.2-1.4 g/kg, i.p.) by the umbilical cord. Caspase-3 activity was measured with fluorescent caspase-3 substrate using an optical imaging system. Hypoxia was induced by the umbilical cord occlusion for 5 minutes. After the initial occlusion and reperfusion of umbilical blood flow, the 2nd-5th occlusions were repeated with reperfusion at 2-5 hours after the first occlusion. The Bcl-2-siRNA or Bax-siRNA was applied into cells of the SC by electropolation. Apoptosis of fetal SC was measured by using the anti-ssDNA antibody. Apoptosis appeared during the occlusion and at 1-5 hrs after the reperfusion of the umbilical cord. Caspase-3 activity was increased transiently during the occlusion and long-lasted largely after the reperfusion. A low dose of Bcl-2 inhibitor did not affect on caspase-3 activity during the occlusion and after the reperfusion. But, high dose of Bcl-2 inhibitor unexpectedly suppressed the caspase-3 activity. Bax inhibitor had no effect on caspase-3 activity. Further, changes in caspase-3 activity of the fetal rats were examined using Bcl-2siRNA and Bax-siRNA to inhibit Bcl-2 and Bax synthesis. Bcl-2-siRNA caused statistically insignificant increases in caspase-3 activity after the 2nd and 3rd reperfusions, whereas Bax-siRNA induced significant decreases. The results suggest that Bcl-2 and Bax may act on an apoptosis cascade in the immature brain during severe hypoxia.