新型磷酸二酯酶4抑制剂氯比普兰改善老年小鼠的认知功能障碍

Chlorbipram,a new phosphodiesterase 4 inhibitor,improves cognition disorders of aged mice

目的探讨新型磷酸二酯酶4抑制剂氯比普兰(chlorbipram)对老年小鼠学习记忆功能障碍的影响。方法 20月龄和6月龄昆明雄性小鼠,分别随机分为溶剂对照组和氯比普兰给药组(0.5 mg·kg-1),连续给药21 d。旷场实验检测穿越格子数、直立次数和粪便粒数;新物体识别实验(NOR)检测识别指数(RI);水迷宫实验检测逃避潜伏期、穿越平台时间、目标象限停留时间及路程和游泳速度。ELISA法测定海马环磷酸腺苷(c AMP)的含量,Western蛋白印迹法检测磷酸化蛋白激酶A(p-PKA)和磷酸化c AMP反应元件结合蛋白(p-CREB)的水平;逆转录PCR(RT-PCR)检测脑源性神经营养因子(BDNF)mRNA的表达。结果旷场实验结果显示,20月龄正常组小鼠粪便粒数明显多于6月龄正常组小鼠(P<0.05),给予氯比普兰后粪便粒数明显减少。NOR实验结果表明,20月龄正常组小鼠RI明显低于6月龄正常组小鼠(P<0.01),给予氯比普兰后RI明显升高(P<0.05),达到6月龄正常组小鼠水平。水迷宫实验结果显示,与6月龄正常组小鼠相比,20月龄正常组小鼠穿越平台次数明显减少(P<0.01),第一次到达平台时间明显延长(P<0.05),目标象限的探索时间和路程显著性下降(P<0.01);给予氯比普兰明显增加穿越平台次数(P<0.01),缩短第一次到达平台时间(P<0.05),延长在目标象限探索的时间和路程(P<0.01)。ELISA分析结果显示,20月龄正常组小鼠海马c AMP水平明显低于6月龄正常组小鼠(P<0.01),氯比普兰能显著增加海马c AMP含量(P<0.01)。Western蛋白印迹结果显示,20月龄正常组小鼠海马内p-PKA和p-CREB的蛋白表达明显低于6月龄正常组小鼠(P<0.01),氯比普兰能够增强p-PKA和p-CREB表达(P<0.01)。RTPCR结果表明,20月龄正常组小鼠海马内BDNF mRNA水平明显低于6月龄正常组小鼠(P<0.01),给予氯比普兰能显著提高BDNF mRNA水平(P<0.01)。结论氯比普兰可以通过影响c AMP水平,上调p-PKA和p-CREB蛋白表达,促进BDNF的转录,从而改善老年小鼠的认知功能障碍。

OBJECTIVE To investigate the effect of the new phosphodiesterase 4 （PDE4） inhibitor chlorbipram on learning and memory in aged mice. METHODS The aged mice were 20-month-old and the young mice were 6-month-old. They were divided into two groups to receive either vehicle or chlorbi- pram 0.5 mg. kg-1 daily for three weeks. The open field test （OFT） was carried out to explore the ambu- lation, rearing and feces. Mice were then subjected to the novel object recognition （NOR） test by record- ing the recognition index of identical and novel objects. The learning and memory ability of these mice was also observed by the Morris water maze （MWM） test to study the time taken to reach the platform, duration and distance in the target quadrant, as well as the entries into the target quadrant and the swim- ming speed. The hippocampus of rats was collected for determination of cyclic adenosine monophos- phate （cAMP） and the expression of phosphorylated protein kinase A （p-PKA）, phosphorylated cAMP response-element binding protein （p-CREB） and brain derived neurotrophic factor （BDNF） mRNA fol- lowed by ELISA, Western blotting and RT-PCR respectively after behavioral tests. ELISA was adopted for the content determination of cAMP and Western blotting for the level of p-PKA and p-CREB. The level of BDNF mRNA was detected by RT-PCR after behavioral tests. RESULTS The OFT showed that the feces of the aged normal control mice far outnumbered those of the young ones（ P〈0.05）, and this effect was reversed obviously by chlorbipram. According to the NOR test, the recognition index in the aged nor- mal control mice was lower than that of the young normal control mice（ P〈0.01 ）, which was significantly improved after chlorbipram treatment（ P〈0.05）. Compared with the young normal control mice, the aged ones took more time to reach the hidden platform in acquisition trial （ P〈0.01 ）, with less exploration time （P〈0.01）, entries（ P〈0.01 ） and distance （P〈0.01） in the target in MWM, while these effects were a- meliorated by chlorbipram treatment. The ELISA analysis demonstrated that the cAMP level of the aged normal control mice was significantly lower than that of the young ones（ P〈0.01 ）. Compared with the young normal control mice, the protein level of p-CREB and p-PKA, and as well as mRNA level of BDNF in the hippocampus of the aged mice, was significantly decreased （P〈0.01）. Chlorbipram elevated cAMP level, protein expression of p-PKA （ P〈0.01 ） and p-CREB（ P〈0.01 ） as well as BDNF mRNA level （ P〈0.01 ） in aged mouse hippocampus. CONCLUSION Chlorbipram appreciably improves the impaired learning and memory behavior in aged mice, which is possibly related to the activation of intracellular cAMP/PKA/CREB signal pathway.