RESUMO
ObjectiveTo investigate the effects of diazoxide pretreatment on expression of phosphatidylinositol 3-kinase(PI3K) mRNA and protein serine/threonine kinase(Akt) mRNA in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R).MethodsThe SD rat myocardial microvascular endothelial cells were cultured.The cells were seeded in 96-well plates ( 100μd/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into 4 groups ( n =25 each):normal control group (group C),H/R group,diazoxide pretreatment group (group DZ) and diazoxide pretreatment + 5-hydroxydecanoate (5-HD,a mitochondrial ATP-sensitive potassium channel blocker) 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 groups DZ and DZ + 5-HD respectively.The cell viability,apoptotic rate and expression of PI3K mRNA and Akt mRNA were detected at the end of reoxygenation.ResultsCompared with group C,the cell viability was significantly decreased,while the apoptotic rate increased and expression of PI3K mRNA and Akt mRNA up-regulated in group H/R (P ＜0.05 or 0.01).Compared with group H/R,the cell viability was significantly increased,while the apoptotic rate decreased and expression of PI3K mRNA and Akt mRNA up-regulated in group DZ (P ＜ 0.05 or 0.01).5-HD could inhibit diazoxide pretreatment-induced changes mentioned above (P ＜ 0.05 or 0.01 ).ConclusionDiazoxide pretreatment can reduce H/R injury by promoting PI3K gene and Akt gene transcription through activation of mitochondrial ATP-sensitive potassium channels in rat myocardial microvascular endothelial cells.
RESUMO
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.
RESUMO
Objective To investigate the effects of diazoxide pretreatment on the expression of NF-κB mRNA and fractalkine (FKN) 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 (100μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1 × 106/ml and randomly divided into4 groups (n = 24 each): Ⅰ normal control group (group C), Ⅱ H/R group, Ⅲ diazoxide pretreatment group (group DZ), Ⅳ 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 100μmol/L were added to the cultured medium 2 h before hypoxia in group DZ and DZ + 5-HD respectively. The cell vitality, apoptotic rate and expression of NF-κB mRNA and FKN mRNA were detected at end of reoxygenation. Results Compared with group C, the cell vitality was significantly decreased, apoptotic rate increased and the expression of NF-κB mRNA and FKN mRNA up-regulated in H/R group. Compared with group H/R, the cell vitality was significantly increased,apoptotic rate decreased and the expression of NF-κB mRNA and FKN mRNA down-regulated in group DZ. 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above. Conclusion Diazoxide pretreatment can reduce H/R injury in rat myocardial microvascular endothelial cells through down-regulating the expression of NFκB and FKN, and the mechanism is related to activation of mitochondrial ATP-sensitive potassium channels.