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目的探讨褪黑素对新生大鼠缺氧缺血性脑损伤(HIBD)的保护作用及其机制。方法 7日龄Wistar大鼠54只结扎左侧颈总动脉,吸入氧氮混合气体55 min制作HIBD模型,随机分成正常对照组、HIBD组和褪黑素治疗组,每组18只。正常对照组既不结扎亦不缺氧,褪黑素治疗组在缺氧缺血前30 min予褪黑素15 mg.kg-1腹腔注射。在缺氧缺血24 h,取每组6只脑组织匀浆用于半胱天冬酶-2、半胱天冬酶-3活性测定,其余每组6只在缺氧缺血72 h取其脑进行石蜡包埋,并进行半胱天冬酶-3、凋亡诱导因子(AIF)及微管相关蛋白-2(MAP-2)的免疫组织化学染色,上述指标用于计算脑梗死体积和海马CA1神经元的丢失,HE染色用于计算脑损伤积分。结果缺氧缺血24 h,HIBD组新生大鼠脑组织半胱天冬酶-2、半胱天冬酶-3活性均明显高于正常对照组(Pa<0.05),给予褪黑素治疗后,半胱天冬酶-2、半胱天冬酶-3活性均明显下降(Pa<0.05);72 h时褪黑素治疗组半胱天冬酶-3和AIF的阳性细胞计数均明显下降(Pa<0.05);褪黑素治疗组脑损伤积分、脑梗死体积、CA1神经元丢失均显著下降(Pa<0.05)。结论褪黑素对HIBD有保护作用,其机制与抑制神经细胞凋亡有关。
Objective To investigate the protective effect of melatonin on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its mechanism. Methods Fifty-four Wistar rats of 7-day-old were ligated with left common carotid artery and inhaled oxygen-nitrogen mixed gas for 55 min to make HIBD model. They were randomly divided into normal control group, HIBD group and melatonin treatment group, with 18 rats in each group. The normal control group neither ligation nor hypoxia, melatonin treatment group 30 minutes before hypoxic-ischemic melatonin 15 mg.kg-1 intraperitoneal injection. Hypoxic-ischemic 24 h, 6 brain tissue homogenates from each group were used for caspase-2 and caspase-3 activity determination, and the remaining 6 in each group were taken 72 h after hypoxia-ischemia The brain was paraffin-embedded and immunohistochemical staining for caspase-3, apoptosis-inducing factor (AIF) and microtubule-associated protein-2 (MAP-2) And loss of hippocampal CA1 neurons, HE staining was used to calculate brain injury scores. Results The activities of caspase-2 and caspase-3 in brain tissue of neonatal rats in HIBD group were significantly higher than those in normal control group (P <0.05) at 24 h of hypoxia-ischemia. After treatment with melatonin , Caspase-2 and caspase-3 activity decreased significantly (Pa <0.05). At 72 h, the positive cell counts of caspase-3 and AIF in melatonin treatment group were significantly decreased (P <0.05). The scores of cerebral injury, the volume of cerebral infarction and the loss of CA1 neurons in melatonin treatment group were significantly decreased (P <0.05). Conclusion Melatonin has a protective effect on HIBD and its mechanism is related to the inhibition of neuronal apoptosis.