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目的于海马微量注射Aβ_(25-35)建立大鼠认知功能障碍模型,研究磷酸二酯酶4(PDE4)抑制剂罗氟司特(Roflumilast)对大鼠认知功能障碍的改善作用及机制。方法 SD大鼠随机分为6组:假手术对照组、Aβ_(25-35)注射组、咯利普兰组(0.5 mg·kg~(-1)·d~(-1))和罗氟司特低剂量组(0.2 mg·kg~(-1)·d~(-1))、中剂量组(0.4 mg·kg~(-1)·d~(-1))、高剂量组(0.8 mg·kg~(-1)·d~(-1))。大鼠双侧海马CA1区微量注射Aβ_(25-35)(10μg)建立认知障功能碍模型。造模24 h后给予相应的药物处理,连续给药21 d。采用旷场实验、水迷宫实验和避暗实验分别检测大鼠的自主活动能力和学习记忆能力。行为学实验结束后利用酶联免疫吸附实验(ELISA)检测海马环磷腺苷(cAMP)水平,采用免疫印迹法(WB)检测海马cAMP应答元件结合蛋白的磷酸化(p-CREB)水平,实时荧光定量PCR(q-PCR)法检测海马脑源性神经营养因子(BDNF)mRNA水平。结果旷场实验中,各组处理对大鼠水平运动和垂直运动得分差异均无统计学意义(P>0.01);水迷宫实验中,与模型组相比,罗氟司特和咯利普兰显能著缩短大鼠的逃避潜伏期并延长大鼠在平台所在象限的停留时间(P<0.05,P<0.01);避暗实验中,模型组大鼠进入暗箱的潜伏期相对于假手术组明显缩短(P<0.01),罗氟司特和咯利普兰则均能显著延长大鼠的进入暗箱的潜伏期(P<0.01);与假手术组相比,模型组大鼠海马cAMP水平显著降低,p-CREB蛋白水平以及BDNF mRNA水平明显下降(P<0.01),而罗氟司特及咯利普兰均能不同程度地改善由Aβ_(25-35)引起的上述改变(P<0.05,P<0.01)。结论罗氟司特能够改善Aβ_(25-35)致痴呆大鼠的学习记忆功能障碍,该作用可能与激活cAMP/CREB信号通路及促进BDNF表达有关。
Objective To establish a rat cognitive impairment model by microinjection of Aβ_ (25-35) into the hippocampus to study the effect and mechanism of Roplumilast, a phosphodiesterase 4 (PDE4) inhibitor, on cognitive dysfunction in rats . Methods SD rats were randomly divided into 6 groups: sham operation control group, Aβ_ (25-35) injection group, rolipram group (0.5 mg · kg -1 · d -1) (0.2 mg · kg -1 · d -1), middle dose (0.4 mg · kg -1 · d -1), high dose (0.8 mg · kg -1 · d -1) mg · kg -1 · d -1). Aβ_ (25-35) (10 μg) microinjection into hippocampal CA1 region of rats was used to establish cognitive impairment model. After 24 h, the rats were given the appropriate drug treatment, continuous administration of 21 d. Open-field test, water maze test and dark-avoidance test were used to detect the autonomic ability and learning and memory ability of rats respectively. Hippocampal cyclic adenosine monophosphate (cAMP) levels were measured by enzyme-linked immunosorbent assay (ELISA) at the end of behavioral experiments. Phosphorylation (c-CREB) level of cAMP response element-binding protein in hippocampus was detected by Western blotting. The levels of brain-derived neurotrophic factor (BDNF) mRNA in hippocampus were detected by q-PCR. Results In open-field test, there was no significant difference in the scores of horizontal motion and vertical motion of rats in each group (P> 0.01). In water maze test, compared with the model group, roflumilast and rolipram (P <0.05, P <0.01). In the dark-avoidance experiment, the latent period of entering the dark box in the model group was significantly shorter than that in the sham-operated group (P < (P <0.01). Both roflumilast and rolipram significantly prolonged the latency to enter the dark box (P <0.01). Compared with the sham operation group, the cAMP level in the hippocampus of the model group was significantly decreased CREB protein and BDNF mRNA levels were significantly decreased (P <0.01), while both roflumilast and rolipram could ameliorate the changes induced by Aβ_ (25-35) (P <0.05, P <0.01) . Conclusion Roflumist can improve learning and memory dysfunction in Aβ 25-35-induced dementia rats, which may be related to the activation of cAMP / CREB signal pathway and the promotion of BDNF expression.