捕食应激对大鼠海马环一磷酸腺苷反应元件结合蛋白表达的影响

Effects of predator stress on the expression of cAMP responsive element binding protein in hippocampus:experiment with rats

目的探讨严重心理应激所致情感行为异常与认知功能受损的神经生物学机制。方法在大鼠捕食应激模型基础上,通过免疫组织化学与蛋白质免疫印迹检测,观测应激大鼠神经细胞核转录因子环一磷酸腺苷(cAMP)反应元件结合蛋白(cREB)、磷酸化 CREB(pCREB)与 CREB 结合蛋白(CBP)表达的分布特点与动态变化规律。结果免疫组化研究显示,捕食应激后12 h 应激大鼠脑组织 CREB 阳性免疫反应信号明显弱于对照组(对照组海马与额叶皮质分别为1.78±0.40和1.18±0.26,应激大鼠分别为0.55±0.13和0.88±0.20,P<0.01),且以海马结构的改变更明显(与额叶皮质相比,P<0.01);pCREB 与 CBP 阳性免疫反应信号则明显高于对照组(P<0.01),而 pCREB表达亦以海马细胞增强得更明显(与额叶皮质相比,P<0.05)。海马组织免疫印迹检测进一步揭示捕食应激后1 h 应激大鼠海马 CREB 即较对照组明显下调(应激大鼠为2.82±0.65,对照组为11.86±2.47,P<0.01),24 h 仍显著低于对照组(应激大鼠为5.12±1.13,对照组为10.56±2.38,P<0.01);CBP 表达则同步明显增多(1、24 h 应激大鼠分别为1.77±0.39和2.44±0.55,对照组分别为1.06±0.24和0.86±0.20,P<0.01);而 pCREB 表达仅于1、12 h 显著增高(1、12 h 应激大鼠分别为2.56±0.59和1.93±0.41,对照组分别为1.04±0.22和0.96±0.21,P<0.01)。结论捕食应激后中枢神经系统,特别是选择性海马结构的 CREB 转录途径的改变,在短暂严重心理应激所致长时程情感行为异常与空间学习和记忆等认知功能受损中可能有重要意义。

Objective To explore the neurobiological basis involved in the pathogenesis of the lasting emotionality and cognitive impairment following severe psychological stress. Methods Ninety-six male Wistar rats were divided randomly into 2 equal groups：group of predator stress （Group PS） put into a cage in the experimental box singly to be exposed to a cat in the box but outside the cage for 23 -57 min until tremor, polypnea, and nares flaring appeared for 6 min so as to establish predator stress models, and control group, put into the cage without non-injurious exposure of cat. 1, 12, and 24 hours later 8 rats from each group were killed with the hippocampus taken out. Western blotting was used to detect the protein expressions of cAMP response element-binding protein （ CREB ）, phosphorylated CREB （ pCREB ） and CREB binding protein （CBP）. Twelve hours after the experiment 24 rats from each group were killed with their brains taken out to obtain serial coronary sections, lmmunohistochemistry was used to detect the positive immunoreactivities of CREB, pCREB, and CBP. Results Immunohistochemistry revealed that the absorbense （A） value of CREB-in the tissues of hippocampus and frontal cortex 12h after the cat exposure of Group PS were 0.55±0. 13 and 0. 88 ±0. 20 respectively, both significantly lower than those of the control group （1.78±0.40 and 1.18 ±0.26 respectively, both P〈0.01）, the A values of. pCREB in the hippocampus and frontal cortex of Group PS were 1.51± 0. 34 and 1.07 ± 0. 24 respectively, both significantly higher than those of the control group （ 0.47 ± 0. 11 and 0.48± 0. 11 respectively, both P 〈 0.01 ）, and the A values of CBP in the hippocampus and frontal cortex of Group los were 1.01 ± 0.23 and 0. 81±0. 18 respectively, both significantly higher than those of the control group （0.52 ±0. 12 and 0. 29 ± 0. 07 respectively, both P 〈 0. 01 ）. Western blotting showed that the CREB protein expression levels 1 h and 24 h after the cat exposure of Group PS were 2. 82±0.65 and 5. 12 ± 1.13 respectively, both significantly lower than those of the control group （ 11.86 ± 2. 47 and 10. 56 ± 2. 38 respectively, both P 〈 0.01 ）, the CBP protein expression levels 1 h and 24 h after the cat exposure of Group PS were 1.77±0. 39 and 2. 44 ±0. 55 respectively, both significantly higher than those of the control group （ 1.06 ＋ 0. 24 and 0. 86 ＋0. 20 respectively, both P 〈0. 01 ）, and the pCREB protein expression levels 1 h and 12 h after the cat exposure of Group PS were 2.56 ＋ 0.59 and 1.93 ＋ 0. 41 respectively, both significantly higher than those of the control group （ 1.04 ＋ 0. 22 and 0. 96 ＋ 0. 21 respectively, both P 〈 0. 01 ）. Conclusion The dysfunction of CREB signaling in the central nervous system, especially in the hippocampal formation after predation stress may play an important role in the long-term neuropsychological sequelae following severe stress.