核转录因子FOXO3a在新生大鼠缺氧缺血性脑损伤神经元凋亡中的作用

Role of the FOXO3a transcription factor in neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage

摘要:

目的：探讨核转录因子FOXO3a在缺氧缺血性脑损伤新生大鼠神经元凋亡中的作用及机制。方法：160只10日龄SD大鼠随机分为缺氧缺血（HI）组和假手术组，HI组在乙醚麻醉下行右侧颈总动脉结扎后缺氧2.5?h；假手术组不结扎颈总动脉，不作缺氧处理。两组在HI后0.5?h、2?h、4?h、8?h和24?h各处死16只大鼠取大脑皮层。应用Western blot定量检测FOXO3a总蛋白、胞核FOXO3a蛋白、胞浆FOXO3a蛋白及Bim蛋白表达。应用TUNEL染色法检测凋亡细胞。结果：与假手术组比较，HI组胞核FOXO3a蛋白于HI后0.5 h表达开始增高，随HI时间延长表达逐渐增高，24?h达高峰（P<0.01）；胞浆FOXO3a蛋白水平在HI后各时间点均低于假手术组（P<0.01）；Bim蛋白在HI后0.5 h表达升高，2?h达高峰，4?h开始降低，至8?h开始恢复至基线水平。TUNEL染色结果显示HI后4 h，凋亡细胞开始增多，24?h达高峰，各时间点凋亡指数均高于假手术组（均P<0.01）。结论：新生大鼠缺氧缺血性脑损伤时FOXO3a自胞浆转位入胞核，诱导靶基因前凋亡蛋白Bim表达，Bim表达上调可能与神经元凋亡有关。

Abstract:

OBJECTIVE: To explore the role and mechanisms of FOXO3a nuclear translocation in neuronal apoptosis after hypoxia-ischemia (HI). METHODS: One hundred and sixty 10-day-old Sprague-Dawly rats were randomly divided into two groups: HI and sham-operated. The right common carotid artery was ligated followed by hypoxia exposure for 2.5 hours in the HI group. The sham-operated group rats were not subjected to carotid artery ligation or hypoxia treatment. Rat cerebral cortex was collected at 0.5, 2, 4, 8 and 24 hours after hypoxia. Western blot was used to detect expression of total FOXO3a protein, pnuclear and cytoplasmic FOXO3a and Bim. TUNEL staining was used to detect apoptotic cells. RESULTS: The nuclear protein of FOXO3a obviously increased from 0.5 to 24 hours after HI in a time-dependent manner compared with the sham-operated group (P<0.01). On the contrary, cytoplasmic protein evidently decreased from 0.5 to 24 hours in the HI group compared with the sham-operated group (P<0.01). Bim protein increased from 0.5 hour, peaked at 2 hours, started to decline at 4 hours (P<0.01), and returned to baseline level at 8 and 24 hours after HI in the HI group compared with the sham-operated group. TUNEL positive cells started to express at 4 hours, and peaked at 24 hours after HI (P<0.01). However, TUNEL positive cells were rarely found in the sham-operated group. CONCLUSIONS: HI induces FOXO3a translocation from cytoplasm to nucleus, and enhances protein expression of its target gene Bim in the neonatal rat brain. The upregulation of Bim expression might be related to neuronal apoptosis