目的 应用海马微量注射Aβ25-35建立的大鼠学习记忆障碍模型,研究新型磷酸二酯酶4抑制剂氯比普兰对学习记忆的改善作用及机制。方法 大鼠随机分为假手术组、Aβ25-35微量注射组(模型组)、氯比普兰低剂量组(0.05 mg·kg-1)、高剂量组(0.15 mg·kg-1)、咯利普兰组及多奈哌齐组。大鼠双侧海马CA1区注射10 μg Aβ25-35造成认知障碍模型,24 h后给予相应的药物处理。采用水迷宫、避暗实验检测大鼠的学习记忆能力,敞箱实验测试大鼠的自主活动能力。行为学实验结束后采用蛋白质印迹法检测海马蛋白激酶A、cAMP应答元件结合蛋白磷酸化水平以及脑源性神经营养因子蛋白水平,利用实时定量聚合酶链反应分析海马组织脑源性神经营养因子 mRNA 水平。结果 水迷宫结果显示,与模型组比较,高剂量氯比普兰、咯利普兰及多奈哌齐给药后均能显著性增加大鼠穿越平台次数及目标象限停留时间百分比。避暗实验中,模型组大鼠的逃避潜伏期显著短于假手术组,氯比普兰和多奈哌齐均能显著延长大鼠的逃避潜伏期。敞箱实验中,无论是垂直方向得分还是水平得分,各组之间均未见显著性差异;蛋白质印迹法结果表明,与模型组比较,氯比普兰低剂量、高剂量、咯利普兰和多奈哌齐组均能显著性逆转Aβ25-35引起的蛋白激酶A、cAMP应答元件结合蛋白磷酸化水平的下降,增加脑源性神经营养因子mRNA及蛋白表达水平。结论 氯比普兰有显著改善阿尔迪海默病动物学习记忆障碍的作用,该效应可能与激活蛋白激酶A/cAMP应答元件结合蛋白信号通路,促进脑源性神经营养因子的表达有关。
Abstract
OBJECTIVE To investigate the effect and mechanism of novel phosphodiesterase 4 inhibitor chlorbipram on learning and memory disorders in Alzheimer′s disease animal model.METHODS The rat model of learning and memory deficits with AD was used by bilateral microinjection of Aβ25-35 into the CA1 region of the hippocampus.Then the rats were randomly divided into six groups:sham-operated group, Aβ25-35 microinjected (model group),chlorbipram treatment(0.05 and 0.15 mg·kg-1)group, rolipram-treated(0.05 mg·kg-1)and donepezil(1.0 mg·kg-1) group. The effect of chlorbipram on memory behavioral performance were evaluated with Morris water maze and step-through passive avoidance test, and the open field test was performed to determine the animal locomotor activity. After the last behavioral performance test, the hippocampus were dissected for further molecular analysis. The protein level of BDNF and the phosphorylation of PKA and CREB were analyzed by Western blotting; the mRNA level of BDNF in the hippocampus was detected by real-time PCR. RESULTS Microinfusion with Aβ25-35 produced impairment of spatial memory in behavioral tests,which was reversed by either PDE4 inhibitor or donepezil administration.Chlorbipram, rolipram and donepezil increased the number of crossing and percent of time in the target quadrant in the Morris water maze probe trial. In the step-through passive avoidance test, the 24 h latency was significantly decreased in rats treated with either chlorbipram or positive control drugs, while no significant difference were shown in total locomotor activity among the groups.Western blot analyses showed that Aβ25-35-microinjection decreased the phosphorylation of PKA and CREB and inhibited the protein expression of BDNF in the hippocampus.Chlorbipram, rolipram and donepezil reversed the reduction of the phosphorylated PKA and CREB induced by Aβ25-35. Moreover, these drugs also enhanced both the mRNA and protein levels of BDNF. CONCLUSION Chlorbipram produces a significant improvement of learning and memory in AD animal, and this effect is due to the mediation of cAMP/PKA/CREB/BDNF signal pathway.
关键词
磷酸二酯酶-4抑制剂 /
氯比普兰 /
阿尔茨海默病 /
脑源性神经营养因子 /
学习记忆
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Key words
phosphodiesterase 4 inhibitor /
chlorbipram /
AD /
brain derived neurotrophic factor /
learning and memory
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中图分类号：
R965
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脚注
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基金
国家自然科学基金-广东联合基金(U1032006);国家“新药创制”科技重大专项(2012ZX09J1211003C);国家自然科学基金资助项目(81373384)
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