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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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Citing This Article：

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).