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目的:研究新生大鼠缺氧缺血脑损伤后海马神经元细胞Caspase-12的表达及参附注射液对其的影响,探讨缺氧缺血性脑损伤(HIBD)后可能的凋亡机制及参附注射液的作用机制。方法:新生7天SD大鼠随机分为假手术组(S组)、生理盐水对照组(C组)、参附治疗组(SF组),每1组均设3、6、12、24 h及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,取右侧海马脑组织匀浆,RT-PCR检测Caspase-12 mRNA的表达,Western-blot检测Caspase-12蛋白表达,TUNEL法检测凋亡形态变化。结果:S组Caspase-12 mRNA表达最低,C组和SF组Caspase-12 mRNA均在12 h达到峰值,SF组Caspase-12 mRNA在6、12、24 h及3天较C组表达明显下降(P<0.01);Caspase-12蛋白表达S组最弱,HI后3hC组和SF组较S组明显增加(P<0.01),24 h达到峰值,7天仍较S组高(P<0.01),SF组表达在12、24 h及3天较C组显著降低(P<0.01);TUNEL法显示HI后3 h C组和SF组脑海马即出现少量凋亡细胞,随时间延长凋亡细胞呈增多趋势,24 h达高峰后开始下降;SF组24 h及3、7天均明显低于C组(P<0.05)。结论:缺氧缺血后脑海马神经元细胞Caspase-12表达增加,细胞凋亡增加,Caspase-12参与了新生大鼠HIBD后神经元损伤的凋亡机制,参附注射液能下调Caspase-12的表达,从而起到对缺氧缺血性脑损伤的保护作用。
Objective: To investigate the expression of Caspase-12 in hippocampal neurons after hypoxic-ischemic brain damage in neonatal rats and the effect of Shenfu injection on it, and to explore the possible mechanism of apoptosis after hypoxic-ischemic brain damage (HIBD) Shen Fu injection mechanism of action. Methods: Seven-day-old SD rats were randomly divided into sham operation group (S group), saline control group (C group) and Shenfu treatment group (SF group). Each group was given 3,6,12,24 h And 3, 7 days and 6 subgroups, 8 in each subgroup. The neonatal rat HIBD model was prepared by the method of Rice. The right hippocampal brain homogenate was used to detect the expression of Caspase-12 mRNA by RT-PCR. Western-blot Caspase-12 protein expression was detected by TUNEL assay. Results: The Caspase-12 mRNA expression in S group was the lowest, Caspase-12 mRNA in C group and SF group reached the peak at 12 h, Caspase-12 mRNA in SF group was significantly lower than that in C group at 6, 12, 24 h and 3 days P <0.01). The expression of Caspase-12 protein in S group was the weaker than that in S group at 3h (P <0.01), and peaked at 24 h (P <0.01) , And the expression of SF in group SF was significantly lower than that in group C at 12, 24 and 3 days (P <0.01). TUNEL assay showed that a small amount of apoptotic cells appeared in hippocampus of group C and SF at 3 h after HI, (P <0.05). After 24 h peak, it began to decline. SF group was significantly lower than that of C group at 24 h and 3, 7 days (P <0.05). Conclusion: After hypoxia-ischemia, the expression of Caspase-12 in hippocampal neurons increased and apoptosis increased. Caspase-12 was involved in the apoptosis of neurons after HIBD in neonatal rats. Shenfu injection could down-regulate Caspase-12 Expression, and thus play a protective effect on hypoxic-ischemic brain damage.