CAJ | 학술논문

线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channel, mitoKATP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mitoKATP通道,二氮嗪(diazoxide,DZ)为选择性的mitoKATP通道开放剂,本实验探讨了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 μmol/L、DZ 30 μmol/L、DZ 100μmol/L和DZ 100μmol/L+5-羟癸酸(5-hydroxydecanoate,5-HD)100μmol/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 μmol/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,100μmol/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.
선립체내막ATP민감갑통도(mitochondrial ATP-sensitive potassium channel, mitoKATP통도)적격활재약물예처리증강신경원대각충손상적내수력과정중발휘착중요작용.신경원내함유봉부적mitoKATP통도,이담진(diazoxide,DZ)위선택성적mitoKATP통도개방제,본실험탐토료DZ예처리능부감소결양복양소치적해마신경원조망,이급DZ여하조공Bcl-2단백화Bax단백적표체.원대배양9～10 d적Sprague-Dawley대서해마신경원수궤분위5조:대조조、DZ 0 μmol/L、DZ 30 μmol/L、DZ 100μmol/L화DZ 100μmol/L+5-간계산(5-hydroxydecanoate,5-HD)100μmol/L.제대조조외,기타사조신경원자결양전3 d개시,매천DZ예처리1 h,련속3 d.체외결양4 h,우복양후24 h,사서람비색법측정해마신경원존활솔,annexin V-FITC류식세포술측정조망솔,Western blot법검측Bcl-2화Bax단백적표체량.결과현시:여대조조비교,결양복양손상현저강저해마신경원적존활솔,승고조망솔.여기타농도비교,100 μmol/L DZ예처리사신경원존활솔승고약15%,이조망솔강저약12%;Bcl-2단백표체증강약60%,Bax단백표체하강근30%.5-HD소제DZ대신경원적보호작용.인차,100μmol/L DZ가통과상조Bcl-2단백표체,강저Bax단백표체,감소결양복양후해마신경원적조망.
Activating mitochondrial ATP-sensitive potassium (mitoKATP) channels is a critical event of pharmacological preconditioning,which can enhance neuronal ability against various insults. mitoKATP 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 μmol/L, DZ 30 μmol/L, DZ 100 μmol/L, DZ 100 μmol/L+5-hydroxydecanoate (5-HD, a selective mitoKATP channel blocker) 100 μmol/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 μmol/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 μmol/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 μmol/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 μmol/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.