二氮嗪预先给药对大鼠心肌微血管内皮细胞缺氧复氧时缺氧诱导因子-1α mRNA和p53mRNA表达的影响

Effects of diazoxide pretreatment on the expression of hypoxia-inducible factor-1α mRNA and p53 mRNA in rat myocardial rnicrovascular endothelial cells exposed to hypoxia-reoxygenation

目的 探讨二氮嚓预先给药对大鼠心肌微血管内皮细胞缺氧复氧时缺氧诱导因子-1α（HIF-1α）mRNA和p53 mRNA表达的影响.方法 培养SD大鼠心肌微血管内皮细胞,以1×106/ml的密度接种于96孔板（100μl/孔）或培养皿（2 ml/皿）,采用随机数字表法,将其随机分为4组（n=24）：正常对照组（C组）不作任何处理,缺氧复氧组（H/R组）、二氮嗪预先给药组（DZ组）、二氮嗪预先给药＋线粒体ATP敏感性钾通道阻断剂5-羟葵酸组（DZ＋5-HD组）均进行缺氧2 h复氧2 h.DZ组和DZ＋5-HD组在缺氧前2 h分别加入100μmol/L二氮嗪、100μmol/L二氮嗪＋100μmol/L 5-羟葵酸.于复氧2h时测定细胞活力、细胞凋亡率、HIF-1α mRNA和p53 mRNA表达水平.结果 与C组比较,H/R组细胞活力降低,细胞凋亡率升高,HIF-1αmRNA和p53 mRNA表达上调（P＜0.01）;与H/R组比较,DZ组细胞活力升高,细胞凋亡率降低,HIF-1αmRNA表达上调,p53 mRNA表达下调（P＜0.05或0.01）;5-羟葵酸可抑制二氮嗪预先给药导致的上述改变（P＜0.05）.结论 二氮嗪预先给药可上调HIF-1和下调p53表达,从而减轻大鼠心肌微血管内皮细胞缺氧复氧损伤,其机制与激活线粒体ATP敏感性钾通道有关.

Objective To investigate the effects of diazoxide pretreatment on the expression of hypoxia-inducible factor-1α （HIF-1α） mRNA and p53 mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation （H/R）.Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates （ 100 μl/hole） or in 6 cm diameter dishes （2 ml/dish） with the density of 1 ×106/ml and randomly divided into 4 groups （ n = 24 each）： normal control group （group C）, H/R group, H/R ＋diazoxide pretreatment group （group DZ） and H/R ＋ diazoxide pretreatment ＋ mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate （5-HD） group （group DZ ＋ 5-HD）. The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L ＋ 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in DZ and DZ ＋ 5-HD groups respectively. The cell vitality, apoptotic rate and expression of HIF-1α mRNA and p53 mRNA were detected at the end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of HIF- 1α mRNA and p53 mRNA up-regulated in H/R group （ P 〈 0.01）. Compared with group H/R, the cell vitality was significantly increased, apoptotic rate decreased, the expression of HIF-1α mRNA up-regulated and the expression of p53 mRNA down-regulated in group DZ （ P 〈 0.05 or 0.01 ）. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above （P 〈 0.05 ）. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through up-regulating the expression of HIF-1α and down-regulating the expression of p53, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.