二氮嗪、环孢菌素A拮抗Aβ142致胆碱能神经元凋亡的研究

山东大学学报(医学版)

二氮嗪、环孢菌素A拮抗Aβ142致
胆碱能神经元凋亡的研究

蒋亮亮¹，付庆喜²，王瑞霞¹，马国诏¹，高建新³，刘克敬³，张镛¹

（1. 山东大学附属省立医院神经内科，济南 250021； 2. 临沂市人民医院神经内科，山东临沂 276003；
3. 山东大学医学院生理研究所，济南 250012）

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-03-16 发布日期:2009-03-16
通讯作者: 马国诏

Diazoxide and Cyclosporine A inhibits cultured primitive rat basal
forebrain cholinergic neuron apoptosis induced by Aβ142

JIANG Liangliang¹, FU Qingxi², WANG Ruixia¹, MA Guozhao¹,
GAO Jianxin³, LIU Kejing³, ZHANG Yong¹

(1. Department of Neurology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China;
2. Linyi People′s Hospital, Linyi 276003, Shandong, China；
3. Department of Physiology, School of Medicine, Shandong University, Jinan 250012, China）

Received:1900-01-01 Revised:1900-01-01 Online:2009-03-16 Published:2009-03-16
Contact: MA Guozhao

摘要/Abstract

摘要： 目的研究ATP敏感性钾离子通道开放剂二氮嗪、环孢菌素A及两者协同对β淀粉样蛋白（Aβ142）致原代培养基底前脑胆碱能神经元凋亡的影响。方法采用2?μmol/L的Aβ142对原代培养大鼠胆碱能神经元进行干预，建立阿尔茨海默病（AD）细胞凋亡模型；采用500?μmol/L二氮嗪(预处理1?h)、20?μmol/L环孢菌素A以及两者协同对其干预，作用不同时间（24、72?h）后置于倒置显微镜下观察细胞形态；采用噻唑蓝（MTT）比色法检测细胞存活率，Western blot法检测细胞内凋亡相关蛋白表达水平的变化。结果① Aβ142作用胆碱能神经元72?h后，Bcl2表达量降低（P＜0.01），细胞色素C、Caspase3、Cleaved caspase3的表达量增加（P＜0.01，P＜0.001，P＜0.01），Bax未见明显变化，Bcl2/Bax比值降低（P＜0.01），细胞形态发生明显损伤性变化，细胞存活率明显降低（P＜0.001）；② 二氮嗪、环孢菌素A以及两者协同作用24?h后Bcl2的表达增加（P＜0.05， P＜0.01，P＜0.01），作用72h能明显增加Bcl2的表达(P＜0.05，P＜0.01，P＜0.01)，并提升Bc2/Bax比率而抑制细胞色素C、Caspase3、Cleaved caspase3表达量(P＜0.05，P＜0.01，P＜0.001)，细胞存活率均增高(P＜0.01，P＜0.001)。结论Aβ142作用胆碱能神经元72?h能促使细胞凋亡，二氮嗪与环孢菌素A及两者协同可以明显拮抗Aβ142致细胞凋亡作用。

关键词: 淀粉样β蛋白, 二氮嗪, 环孢菌素, 胆碱能神经元, ATP敏感性钾离子通道, 大鼠,Wistar

Abstract:

To investigate the effects of diazoxide (ATPsensitive potassium channel opener) and cyclosporine A and their combination on apoptosis induced by betaAmyloid protein (Aβ142) on cultured primitive rat basal forebrain cholinergic neurons. MethodsThe cell apoptosis model was induced by Aβ142(2?μmmol/L), and then 500?μmol/L diazoxide, 20?μmol/L cyclosporine A and their combination were used to interfere with it. Cell morphology was observed by an inverted microscope,changes of cell apoptosis were determined by MTT, and expressions of target proteins (bcl2, bax, cytochrome C, caspasce3 and Cleaved Caspase3) were determined by Western blot when the neurons were interfered with by the drugs for different times (24, 72?h). Results ① Being exposed to Aβ142 for 72?h, cell activity remarkably decreased(P＜0.001), expression of Bcl2 decreased (P＜0.01) and expressions of cytochrome C, caspasce3 and Cleaved Caspase3 increased(P＜0.01, P＜0.001， P＜0.01). Expression of Bax had no change but the value of Bcl2/Bax decreased(P＜0.01). ② Expression of Bcl2 increased when the cell apoptosis model was exposed to diazoxide, cyclosporine A and their combination for 24?h（P＜0.05， P＜0.01，　P＜0.01). When the cell apoptosis model was exposed to diazoxide, cyclosporine A and their combination for 72?h, expression of Bcl2 obviously increased (P＜0.05, P＜0.01, P＜0.001), expressions of Cytochrome C, Caspasce3 and Cleaved Caspase3 decreased(P＜0.05, P＜0.01, P＜0.001), and cell activity increased(P＜0.01, P＜0.001). ConclusionBeing exposed to Aβ142 for 72?h，　neuron apoptosis occurs. Diazoxide and cyclosporine A and their combination could protect neurons from apoptosis induced by Aβ142.

Key words: Amyloid betaprotein, Diazoxide, Cyclosporine, Cholinergic neuron, ATPsensitive potassium channel, Rats,Wistar

中图分类号:

R749.16

蒋亮亮1 ，付庆喜2 ，王瑞霞1 ，马国诏1 ，高建新3 ，刘克敬3 ，张镛1
. 二氮嗪、环孢菌素A拮抗Aβ142致
胆碱能神经元凋亡的研究[J]. 山东大学学报(医学版), 2009, 47(03): 23-29.

JIANG Liangliang¹, FU Qingxi², WANG Ruixia¹, MA Guozhao¹,
GAO Jianxin³, LIU Kejing³, ZHANG Yong¹

. Diazoxide and Cyclosporine A inhibits cultured primitive rat basal
forebrain cholinergic neuron apoptosis induced by Aβ142 [J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2009, 47(03): 23-29.

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