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目的探讨神经元的抗凋亡保护机制,评价环磷酸腺苷(cAMP)反应元件结合蛋白(CREB)、神经生长因子(NGF)对神经元损伤后的保护作用。方法7日龄Sprague-Dawley新生大鼠随机分为假手术组、缺氧缺血性脑损伤(HIBD)未治疗组、小干扰RNA(siRNA)治疗组、siRNA加NGF治疗组。按照改良的Rice法制备HIBD再灌注模型,各治疗组于再灌注后开始予1次siRNA干预治疗(10mg/kg);siRNA加NGF组予NGF(15μg/kg),1次/d。于治疗的不同时间通过免疫组织化学和Western blot法检测磷酸化的CREB、NGF在仔鼠HIBD再灌注后海马、丘脑神经元的表达变化。通过Thionine染色检测神经元凋亡。结果HIBD未治疗组3h仔鼠右侧海马CA1区磷酸化的CREB(p-CREB)表达达高峰,7d后降至假手术对照水平。3hNGF表达开始增加,12h持续达高峰,7d后仍明显高于假手术组(P<0.05)。siRNA治疗组p-CREB与NGF表达随时间逐渐下降。HIBD再灌注未治疗组24h右侧海马CA1区已有明显的凋亡,7d神经元凋亡与假手术组相比无统计意义。siRNA治疗组神经元有大量的丢失;而siRNA加NGF治疗组神经元与假手术组比较无明显丢失。结论CREB经信号转导调节大鼠NGF的表达,从而促进其神经元损伤后修复,减少神经元死亡。
Objective To investigate the anti-apoptotic mechanism of neurons and to evaluate the protective effect of cAMP response element-binding protein (CREB) and nerve growth factor (NGF) on neurons. Methods 7-day-old Sprague-Dawley rats were randomly divided into sham operation group, HIBD untreated group, small interfering RNA (siRNA) treatment group and siRNA plus NGF treatment group. The model of HIBD reperfusion was established according to the modified Rice method. Each treatment group was treated with siRNA (10mg / kg) once after reperfusion. NGF (15μg / kg) and NGF (1μg / kg) The expression of phosphorylated CREB and NGF in the hippocampus and thalamic neurons after HIBD reperfusion were detected by immunohistochemistry and Western blot at different time points. Neuronal apoptosis was detected by Thionine staining. Results The expression of phosphorylated CREB (p-CREB) in the hippocampal CA1 region in the right hippocampus of HIBD untreated group reached its peak at 3h, and dropped to the sham-operated control level after 7 days. 3hNGF expression began to increase, peaked at 12h, and remained significantly higher than sham-operated group after 7 days (P <0.05). The expression of p-CREB and NGF in siRNA-treated group decreased gradually with time. There was obvious apoptosis in hippocampal CA1 region of right hippocampus in HIBD reperfusion untreated group at the 7th day, and there was no significant difference between the 7th day and the sham operation group. siRNA treatment group neurons have a lot of loss; and siRNA plus NGF treatment group neurons and sham operation group no significant loss. CONCLUSIONS: CREB regulates the expression of NGF in rats through signal transduction, thereby promoting neuronal injury repair and reducing neuronal death.