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目的:研究新生大鼠脑缺氧缺血后不同时间点血红素加氧酶-1(hemeoxygen-ase,HO-1)、Caspase-3在海马神经元中的动态变化及纳洛酮注射液的干预作用,进一步探讨HO-1、Caspase-3在新生大鼠缺氧缺血后神经损伤过程中的可能机制及纳洛酮注射液对神经系统保护的作用。方法:新生7天龄SD大鼠随机分为3组:假手术组(S)、生理盐水对照组(C)、纳洛酮干预组(N),每组按照观测的时间点不同分为3、6、12、24 h和3、7天6个亚组,每个亚组8只,用Rice法制备新生大鼠HIBD模型。在每个时间点将大鼠断头取右侧海马脑组织匀浆,用Western blot方法测不同时间点HO-1、Caspase-3动态变化;用TUNEL染色法检测相应时间点脑海马CA1区细胞凋亡。结果:①Western蛋白印迹S组海马HO-1表达很弱,各时间点表达无差异(P>0.05);C组和N组右侧海马HO-1在HI后3h即明显增加(P<0.01),HI后24 h海马HO-1蛋白表达达到峰值,3天后明显下降,7天接近S组,但仍较正常偏高(P<0.01);N组在12、24 h和3、7天海马HO-1表达均较C组高(P<0.01)。②在HI后3 h,C组和N组右侧海马Caspase-3即明显增加(P<0.01),HI后24 h海马Caspase-3蛋白表达达到峰值,3天后明显下降,7天接近S组(P=0.519);N组在12、24 h、3天海马HO-1表达均较C组低(P<0.01)。③Tunel显示S组各时间点右侧海马CA1区仅见少量凋亡细胞,HI后3 h C组和N组右侧海马神经元凋亡细胞数即明显增加(P<0.01),24 h达到高峰,3天开始下降,7天时仍高于S组(P<0.01);N组凋亡数在24 h、3、7天这3个时间点上均较C组明显下降(P<0.01)。结论:HI后脑海马组织细胞中HO-1蛋白、Caspase-3表达均是增加的,两者表达趋势相一致,在时间上吻合,表明HO-1、Caspase-3参与了新生大鼠HI后细胞凋亡的病理过程;纳洛酮注射液能够上调HO-1的表达,抑制Caspase-3活化,减少神经元凋亡,从而起到对HIBD新生大鼠保护作用。
Objective: To investigate the dynamic changes of hemeoxygen-ase (HO-1) and Caspase-3 in hippocampal neurons of neonatal rats after hypoxia-ischemia and the effects of naloxone injection To explore the possible mechanism of HO-1 and Caspase-3 in the process of neural injury after hypoxic-ischemic brain damage in neonatal rats and the effect of naloxone on neuroprotection. Methods: Newborn 7-day-old SD rats were randomly divided into 3 groups: sham operation group (S), saline control group (C) and naloxone intervention group (N), and each group was divided into three groups according to the observed time points , 6,12,24 h and 3,7 days 6 subgroups, 8 in each subgroup. The neonatal rat HIBD model was prepared by the method of Rice. At each time point, the rats were decapitated and the right hippocampus was homogenized. The dynamic changes of HO-1 and Caspase-3 at different time points were detected by Western blot. The expression of Caspase-3 in hippocampus was detected by TUNEL staining Apoptosis. Results: ① Western blotting showed that the expression of HO-1 in hippocampus of S group was weak and there was no difference at each time point (P> 0.05); HO-1 of right hippocampus in C group and N group increased significantly 3h after HI (P <0.01) The protein expression of HO-1 in hippocampus peaked at 24 h after HI, and decreased significantly after 3 days. It was close to S group at 7 days but still higher than normal (P <0.01) HO-1 expression was higher than C group (P <0.01). ② At 3 h after HI, the expression of Caspase-3 in the right hippocampus of C and N groups was significantly increased (P <0.01), and the expression of Caspase-3 protein in hippocampus peaked at 24 h after HI and decreased significantly after 3 days (P = 0.519). The expression of HO-1 in hippocampus of group N at 12, 24 and 3 days was lower than that of group C (P <0.01). ③Tunel showed that only a small amount of apoptotic cells were observed in hippocampal CA1 region of right temporal hippocampus in group S at each time point. The numbers of apoptotic neurons in right hippocampal neurons increased significantly (P <0.01) at 3 h after HI in HI group and peaked at 24 h (P <0.01). The number of apoptosis in N group was significantly lower than that in C group at 24 h, 3 and 7 days (P <0.01) at 3 days. Conclusion: The expression of HO-1 protein and Caspase-3 in hippocampal hippocampus tissue of hippocampus of HI rats are increased, and the expression trend of HO-1 and Caspase-3 are in good agreement with each other. Naloxone injection could up-regulate the expression of HO-1, inhibit the activation of Caspase-3, and reduce the apoptosis of neurons, which play a protective role in neonatal rats with HIBD.