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二氮嗪预处理上调Bcl--2/Bax蛋白比值减少缺氧复氧所致体外培养的海马神经元凋亡

刘荣国, 汪炜健, 宋娜, 陈彦青, 李立环

福建省立医院麻醉科.福建,福州 350001；温州医学院第一附属医院麻醉科.浙江,温州 325000；医科院阜外心血管病医院麻醉科.北京 100037

摘要

线粒体内膜ATP敏感钾通道(mitochondrial ATP--sensitive potassium channel, mitoK_(ATP)通道)的激活在药物预处理增强神经元对各种损伤的耐受力过程中发挥着重要作用。神经元内含有丰富的mitoK_(ATP)通道，二氮嗪(diazoxide, DZ)为选择性的mitoK_(ATP)通道开放剂，该实验探讨了DZ预处理能否减少缺氧复氧所致的海马神经元凋亡，以及DZ如何调控Bcl--2蛋白和Bax蛋白的表达。原代培养9---10 d的Sprague--Dawley大鼠海马神经元随机分为5组：对照组、DZ 0 #mu#mol/L、DZ 30 #mu#mol/L、DZ 100 #mu#mol/L和DZ 100 #mu#mol/L+5--羟癸酸(5--hydroxydecanoate, 5--HD) 100 #mu#mol/L。除对照组外，其他四组神经元自缺氧前3 d开始，每天DZ预处理1 h，连续3 d。体外缺氧4 h，于复氧后24 h，四唑蓝比色法测定海马神经元存活率，annexin V--FITC流式细胞术测定凋亡率，Western blot法检测Bcl--2和Bax蛋白的表达量。结果显示：与对照组比较，缺氧复氧损伤显著降低海马神经元的存活率，升高凋亡率。与其他浓度比较，100 #mu#mol/L DZ预处理使神经元存活率升高约15%，而凋亡率降低约12%；Bcl--2蛋白表达增强约60%，Bax蛋白表达下降近30%。5--HD消除DZ对神经元的保护作用。因此，100 #mu#mol/L DZ可通过上调Bcl--2蛋白表达，降低Bax蛋白表达，减少缺氧复氧后海马神经元的凋亡。

关键词： 二氮嗪; 凋亡; 细胞缺氧; Bcl-2蛋白; 海马

Diazoxide preconditioning alleviates apoptosis of hippocampal neurons induced by anoxia--reoxygenation {sl in vitro} through up--regulation of Bcl--2/Bax protein ratio

Liu Rongguo, Wang Weijian, Song Na, Chen Yanqing, Li Lihuan

Department of Anesthesiology, Fujian Provincial Hospital.Fuzhou 350001,Fujian；China；Department of Anesthesiology, Cardiovascular Institute,Fuwai Hospital Chinese Academy of Medical Sciences.Beijing 100037

Abstract

Activating mitochondrial ATP-sensitive potassium (mitoK_(ATP)) channels is a critical event of pharmacological preconditioning,which can enhance neuronal ability against various insults. mitoK_(ATP) channels are abundant in neurons and can be selectively opened by diazoxide (DZ). The aim of this study was to determine whether DZ could restrain neuronal apoptosis induced by anoxia-reoxygenation and to reveal the effect of DZ preconditioning on the expressions of Bcl-2 and Bax proteins in cultured hippocampal neurons. Cultured for 9--10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control, DZ 0 #mu#mol/L, DZ 30 #mu#mol/L, DZ 100 #mu#mol/L, DZ 100 #mu#mol/L+5-hydroxydecanoate (5-HD, a selective mitoK_(ATP) channel blocker) 100 #mu#mol/L. Prior to oxygen deprivation, the hippocampal neurons except those in the control group were treated with DZ or DZ+5-HD for 1 h per day and this treatment persisted for 3 d. Thereafter, neurons were subjected to anoxia for 4 h and followed by reoxygenation.At 24 h of reoxygenation the neuronal survival rates were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Bcl-2 and Bax proteins were detected with immunocytochemistry and evaluated by Western blot. Anoxia-reoxygenation injury reduced the survival rates and increased apoptotic rates significantly. In comparison with those in other groups, the survival rate in DZ 100 #mu#mol/L group was increased by about 15%, whereas the apoptotic rate was decreased by almost 12% simultaneously. 5-HD could abolish the neuroprotection afforded by 100 #mu#mol/L DZ. Bcl-2 and Bax proteins in the control normoxic neurons were both expressed slightly, while anoxia-reoxygenation led to high expression of Bax protein. The administration of 100 #mu#mol/L DZ enhanced the expression of Bcl-2 protein by nearly 60%, whereas Bax protein was reduced by approximately 30%. Lower concentrations of DZ had no detectable effects on the expressions of Bcl-2 and Bax proteins. However, beneficial effects of DZ on the expressions of Bcl-2 and Bax proteins were reversed after the co-treatment with 5-HD. In conclusion, 100 #mu#mol/L DZ prevented cultured hippocampal neurons from apoptosis induced by anoxia-reoxygenation possibly through up-regulating the expression of Bcl-2 protein and down-regulating the expression of Bax protein.

Key words: Diazoxide;Apoptosis;cell hypoxia;proto-oncogene protein c-Bcl-2;hippocampus

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刘荣国, 汪炜健, 宋娜, 陈彦青, 李立环. 二氮嗪预处理上调Bcl--2/Bax蛋白比值减少缺氧复氧所致体外培养的海马神经元凋亡[J]. 生理学报 2006; 58 (4): .

Liu Rongguo, Wang Weijian, Song Na, Chen Yanqing, Li Lihuan. Diazoxide preconditioning alleviates apoptosis of hippocampal neurons induced by anoxia--reoxygenation {sl in vitro} through up--regulation of Bcl--2/Bax protein ratio. Acta Physiol Sin 2006; 58 (4): (in Chinese with English abstract).