Diazoxide preconditioning alleviates apoptosis of hippocampal neurons induced by anoxia-reoxygenation in vitro through up-regulation of Bcl-2/Bax protein ratio.
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 mumol/L, DZ 30 mumol/L, DZ 100 mumol/L, DZ 100 mumol/L+5-hydroxydecanoate (5-HD, a selective mitoK(ATP) channel blocker) 100 mumol/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 mumol/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 mumol/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 mumol/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 mumol/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.
Liu
Rong-Guo
RG
Department of Anesthesiology, Fujian Provincial Hospital, Fuzhou, China.
Wang
Wei-Jian
WJ
Song
Na
N
Chen
Yan-Qing
YQ
Li
Li-Huan
LH
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia; D003981:Diazoxide / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology*; D019194:Ischemic Preconditioning / Q000379:methods*; D009474:Neurons / Q000166:cytology; D061251:Primary Cell Culture; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000517:prevention & control*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
C494828:Bax protein, rat; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D003981:Diazoxide