褪黑素对异氟烷诱导的发育神经元凋亡及细胞色素C和半胱氨酸蛋白酶-3释放的影响

Effect of melatonin on isoflurane-induced developing neuron apoptosis and release of Cyt-C and caspase-3

目的探讨褪黑素(MT)对全身麻醉药诱导发育神经元凋亡的改善作用及可能的作用机制。方法出生后7日龄(P7)Sprague-Dawley幼鼠40只,分入异氟烷组(予异氟烷吸入麻醉,吸入体积分数为0.013)、异氟烷低剂量MT组(异氟烷麻醉前15min予幼鼠腹腔内注射MT5mg/kg,麻醉2h后重复注射1次同剂量MT)、异氟烷高剂量MT组(两次注射MT的剂量为20mg/kg)和空白组(吸入空气,持续3h),每组10只。观察麻醉结束后各组幼鼠大脑海马CA1区神经元细胞色素C(Cyt-C)、半胱氨酸蛋白酶-3(Caspase-3)的免疫组织化学染色结果,并计算终端dUTP末端标记法(TUNEL)测定的凋亡指数(AI)。结果空白组幼鼠海马CA1区神经元细胞质基本未见Cyt-C和Caspase-3阳性染色,神经元存在少量点状凋亡。异氟烷组Cyt-C和Caspase-3阳性染色明显增加,凋亡神经元数目明显增多,Cyt-C和Caspase-3光密度值、AI均显著高于空白组(P值均<0.05)。异氟烷低剂量MT组和异氟烷高剂量MT组的Cyt-C和Caspase-3光密度值、AI均显著低于异氟烷组(P值均<0.05),但仍显著高于空白组(P值均<0.05);异氟烷低剂量MT组与异氟烷高剂量MT组间的差异均无统计学意义(P值均>0.05)。结论 MT对全身麻醉药诱导的发育神经元凋亡具有改善作用,其作用机制可能与抑制线粒体途径中Cyt-C的胞质释放,减少下游Caspase-3的活化,进而减少发育神经元的异常凋亡有关。

Objective To investigate the protective effect of melatonin （MT） on isoflurane-induced apoptosis of developing neurons and the potential mitochondrial mechanism. Methods Seven-day-old （PT） Sprague-Dawley rats were randomly divided into 4 groups, including isoflurane group （volume fraction of inhaled isoflurane was 0. 013）, low dose MT group （MT 5 mg/kg were administered by intraperitoneal injection 15 rain before isoflurane application and repeated after 2 h）, high dose MT group （MT 20 mg/kg was intraperitoneally injected twice） and control group （air exposure for 3 h）. In hippocampal CA1 region, the expression of cytochrome C （Cyt-C） and caspase-3 was observed by immunohistochemical staining, and apoptotic index （AI） was detected by terminal dUTP nick end labeling （TUNEL） after anesthesia. Results In control group, positive expression of Cyt-C and caspase-3 was rarely observed in the cytoplasm and neuron apoptosis was scattered in hippocampal CA1 region. In isoflurane group, positive expression of Cyt-C and caspase-3 and amounts of apoptotic neurons were significantly increased. Optical density values of Cyt-C and caspase-3 and AI in isoflurane group were ＇significantly higher than those in control group （all P〈0.05）. Optical density values of Cyt-C and caspase-3 and AI in both MT groups were significantly lower than those in isoflurane group （all P〈0.05）, while those indicators in both MT groups were significantly higher than control group （all P〈0.05）. There is no significant difference in optical density value or AI between the two MT groups （all P〈0.05）. Conclusion MT can ameliorate isoflurane-induced neuron apoptosis in developing brain, which may be related to the inhibition of cytosolic release of Cyt-C in the mitochondria and reduce of caspase-3 activation in the downstream pathway.