| Objective:To explore the protection effects and it's mechanisms of mitochondrial ATP-sensitive potassium channel mediated human adenocarcinoma of lung A549 cells from human typeⅡalveolar epithelial cells apoptosis induced by hyperoxia. Methods:Human adenocarcinoma of lung A549 cells from human typeⅡalveolar epithelial cells was used as the target cell. The A549 cells were passaged and cultured in vitro. The attacking factor of oxygen was mixed by O2 (90%) and CO2 (5%) at a velocity of 3L/min. The experiment were divided into control group, hyperoxia group, hyperoxia and treated with diazoxide (the concentration finally is 100mol/L) group. Then the change of morphology were observed under inverted microscope, and the cell viability were measured by cell counting kit-8 (CCK-8) method, and cell apoptosis was detected by flow cytometry (FC). The expression of Omi/HtrA2 were detected by immunohistochemical methods. The mitochondria membrane potential were observed under confocal microscopy. The changes of the ultrastructure of cells were observed by transmission electron microscope (TEM). Results:(1) Under inverted microscopy, A549 cells from the control group were significantly increased, sticked to each other tightly and grew very quickly. Their adhesion were better, multy-angle oblate, showed like paving stones, many cells were in division phase. Compared with the control group, the changes in morphology of A549 were most serious and obvious in the hyperoxia group, the cells grew slowly, the mount decreased, form of the cells changed from typical multy-angle oblate to round or ellipse. Gap between cells were increased,and contour enhanced. There was much cell debris in accrescent intercellular space.But, the changes in morphology of A549 were obviously improved in the group of pretreatment with diazoxide. (2)The cell viability were decreased in hyperoxia group (0.13±0.02). But compared with the hyperoxia group, the cell viability was increased in group of pretreatment with diazoxide (0.21±0.04), but not to the same as that of thecontrol group (0.34±0.03) (P<0.01). (3)Compared with the control group (0.40%±0.10%), the apoptosis of A549 cells was significantly increased in the hyperoxia group (6.54%±0.48%); But compared with the hyperoxia group, the apoptosis of A549 cells was obviously decreased in group of pretreatment with diazoxide (2.36%±0.51%) (P<0.01). (4) Compared with controls group (15.26±2.80), the expression of Omi/HtrA2 were significantly increased in the hyperoxia group (44.94±4.47); But compared with the hyperoxia group, the expression of Omi/HtrA2 were significantly decreased in group of pretreatment with diazoxide (28.22±1.07) (P<0.01). (5)Compared with the control group (1.92±0.11), the JC-1 fluorescence ratios were obviously decreased in the hyperoxia group (1.00±0.08). But compared with the hyperoxia group, the JC-1 fluorescence ratios were significantly increased in group of pretreatment with diazoxide (1.57±0.08) (P<0.01). (6)The A549 cells have complete structure in the control group. Nuclei appeare oval. Large number of multivesicular body can be seen in the cytoplasm. The mitochondria structure are not tumidness. Compare with control group, nuclei appeared roundness; A large number of mitochondria swell; Electron density and mitochondrialcristae decrease in the hyperoxia group. In group of pretreatment with diazoxide, nuclei appeared small and roundness; Electron density are higher; The mitochondria seemed like vacuoles and mitochondrialcristae decrease. Conclusion:The diazoxide Diazoxide could reduce the expression of Omi/HtrA2, and stabilize mitochondria, reduce apoptosis of A549 cells to relieve the A549 cell's injury induced by hyperoxia.
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