咖啡因对苯妥英诱导的小脑颗粒神经元凋亡的保护作用及机制  

Protective effects of caffeine on apoptosis of cerebellar granule neurons induced by diphenylhydantoin

作　　者：赵灵芝[1] 苏兴文[2] 银巍[2] 江伟健[2] 黄亦俊[2] 邱鹏新[2] 颜光美[2] 

机构地区：[1]中山大学药学院药理毒理实验室,广东广州510089 [2]中山大学基础医学院药理教研室,广东广州510089

出　　处：《中国药理学通报》2004年第12期1370-1374,共5页Chinese Pharmacological Bulletin

基　　金：广东省卫生厅(B2004026);广东省自然科学基金(No.04300307);国家杰出青年科学基金(No39625022)资助项目

摘　　要：目的　观察咖啡因 (caffeine)对苯妥英 (diphenylhy dantoin ,DPH) 10 0 μmol·L-1处理的大鼠小脑颗粒神经元(cerebellargranularneurons ,CGNs)存活率的影响 ,并探讨其作用机制。方法　体外培养 8d的CGNs,同时给予 10 0μmol·L-1苯妥英和 1 2 5～ 2 0mmol·L-1咖啡因 ,4 8h后行凋亡分析 ;采用dantrolene(2 0 μmol·L-1)、2APB (5 0 μmol·L-1)、nifedipine(10 0 μmol·L-1)和nimodipine(10 0 μmol·L-1)、MK80 1(4μmol·L-1)、KN93(1μmol·L-1)以及MEK1抑制剂PD980 5 9(5 0 μmol·L-1)分别预先孵育 30min ,再与10mmol·L-1咖啡因和 10 0 μmol·L-1苯妥英共孵育 4 8h ,测定CGNs存活率 ,观察咖啡因的作用与 [Ca2 + ]i 的关系 ;Westernblot法检测咖啡因对磷酸化c Jun和磷酸化ERK水平的影响。结果　① 1 2 5～ 2 0mmol·L-1咖啡因可浓度依赖性抑制 10 0 μmol·L-1苯妥英引起的CGNs凋亡 ,显著提高CGNs存活率 ;②dantrolene、2APB、nifedipine和nimodipine、KN93、MK80 1和PD980 5 9均不能取消 10mmol·L-1咖啡因对 10 0 μmol·L-1苯妥英引起的CGNs凋亡的保护作用。③咖啡因可明显抑制苯妥英诱导CGNs中c Jun磷酸化水平的升高 ,但不影响被苯妥英抑制的ERK的活性。结论　一定浓度的咖啡因可保护苯?Aim To investigate the effects of caffeine on apoptosis of cerebellar granule neurons induced by diphenylhydantoin(DPH) and its possible mechanisms. Methods Cerebellar granule neurons(CGNs), primarily cultured for 8 days, were co-incubated with 100 μmol·L -1 DPH and caffeine of various concentrations for 48 h and then submitted to apoptotic analysis. CGNs, bpre-treated treated with 100 μmol·L -1 DPH and 10 mmol·L -1 caffeine for 48 h, were incubated with dantrolene,2APB,nifedipine and nimodipine, MK801, KN93 or PD98059 for 30 min,respectively, and then cell viability assays were performed to study the relationship of caffeine protection with[Ca 2+] i. Western blotting was further emplyed to compare the levels of phosphor-ERK or phosphor-c-Jun in DPH-treated CGNs plus with or without caffeine to find the possible signal transduction pathway likely involved in the protection of caffeine on CGNs treated with DPH. Results Caffeine from the concentration of 1.25 to 20 mmol·L -1 displayed concentration-dependent protective effects which couldn’t be abolished either by inhibitors of internal Ca 2+ stores or by blockers of Ca 2+ influx. However, caffeine markedly suppressed the phosphorylation of c-Jun, which could be significantly elevated in CGNs treated wih DPH, but demonstrate little to no effects on phosphor-ERK which could benotably inhibited in CGNs in the condition of incubation with DPH.Conclusion Caffeine, which was reported to be an agonist of rynodine receptor, blocks the DPH-induced apoptosis of CGNs by calcium and ERK-independent mechanisms, but most likely through a way of c-Jun phosphorylation inhibition.

关 键 词：咖啡因 苯妥因 小脑颗粒神经元 凋亡 胞外信号反应激酶(ERK) C-JUN 

分 类 号：R-332[医药卫生] R322.81