罗氟司特对Aβ_(25-35)致大鼠认知功能障碍的改善作用及机制研究

Roflumilast Ameliorated Cognitive Impairment in AD Rats Induced by Aβ_(25-35) and Its MechanIsms Involved

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 investigate the effect and mechanism of PDE4 inhibitor roflumilast on cognitive impairment in Alzheimer＇s disease animal model. METHODS The Alzheimer＇s disease model was produced by microinjection of Aβ_（25-35）（ 10 μg） into the bilateral hippocampal CA1 region of male rats. Then the rats were randomly divided into six groups： sham-operated control group,Aβ_（25-35） microinjected group,rolipram treated（ 0.5 mg·kg^（-1）·d^（-1）） group,roflumilast treated group with different dose（ 0.2,0.4 and 0.8mg·kg^（-1）·d^（-1））.Treatments was given（ ig） 24 h after surgery operation for consecutive 21 days. Open field test,Morris water maze and passive avoidance test were used to determine the effect of roflumilast on memory behavior performances. The level of cAMP in hippocampus was analyzed by ELISA; the protein level of phosphorylation of CREB（ p-CREB） was analyzed by Western blotting; the mRNA level of BDNF was measured by real time q-PCR. RESULTS In Morris water maze test,roflumilast and rolipram shorten escape latency and increased time spend in the target quadrant of rats（ P〈0. 05,P〈0. 01）; in the passive avoidance test,the passive avoidance latency was significantly decreased in rats treated with roflumilast and rolipram（ P〈0.01）,while theses effects were based on without any influence on locomotor activity of rats. Results of biochemical analysis revealed that the hippocampus level of cAMP,protein level of p-CREB and mRNA level of BDNF were significantly decreased（ P〈0.01） induced by microinjection of Aβ_（25-35）. Roflumilast and rolipram ameliorated those changes induced by Aβ_（25-35）（ P〈0.05,P〈0.01）. CONCLUSION The PDE4 inhibitor roflumilast produces a significant improvement of cognitive function in AD animal induced by Aβ_（25-35） infusion,which related to its mediation of cAMP/CREB/BDNF signal pathway.