Effect Of Hypoxic Postconditioning On Alveolar Epithelial Type â…¡ Cell Mitochondrial Apoptotic Pathway

Objective:To investigate the effects of hypoxic postconditioning on apoptosis of typeⅡalveolar epithelial cells mitochondrial pathway and provide the new experimental evidence in the protection measures of acute lung injury.Methods:The third generations of subculture human typeⅡalveolar epithelial cells(A 549 cell) were separated into 3 groups randomly. They were normoxia group, hypoxia-reoxygenation group and hypoxic postconditioning group, respectively.①normoxia group: the cells were cultured to confluence in six 6-well culture plates, incubated in normoxic conditions (5%CO2,95% air at 37℃) for 24 hours.②hypoxia-reoxygenation group (H/R group):the cells were cultured to confluence in six 6-well culture plates,subjected to stringent hypoxia (5% CO2,95% N2 at 37℃) for 12hours, and returned to normoxic conditions (5% CO2,95% air at 37℃) for 12hours.③hypoxic postconditioning group (H-PostC group):hypoxic postconditioning (5R/5H, three times) between the end of hypoxic(5% CO2,95% N2 at 37℃)for 12h and the beginning of reoxygenation(5% CO2, 95% air at 37℃) followed by reoxygenation for 12h. Inverted phase contrast microscope, Flow cytometry and RT-PCR method were used to observe cell morphological change, Apoptosis rate and Bax/Bcl-2 gene expressions, respectively.Results:①Cell morphology changes:The normoxia group displayed:the cells were spindle shape and bright, sticked to the bottle firmly, cell suspension could not been found. The hypoxia-reoxygenation group displayed:cells became round, intercellular space became broad, there were cell lysozyme, floating cells and cell debris in the culture medium. The hypoxic postconditioning group displayed:a few of the cells became round and the intercellular space became a little broad.②Cell apoptosis rate:There was significant statistical difference among the three groups(P<0.05). Compared with normoxia group(0.3000±0.1276), it significantly increased in H/R group (7.2517±1.2648) (P<0.05). And compared with H/R group, that in H-PostC group (4.1783±1.0364) was significantly decreased (P<0.05).③Bax gene expression:There was significant statistical difference among the three groups(P<0.05). Compared with normoxia group (12.4733±1.0657), it significantly increased in H/R group (22.6800±0.3628) (P<0.05). And compared with H/R group, H-PostC group (16.9583±0.6151) significantly decreased (P<0.05).④Bcl-2 gene expression:There was significant statistical differences among three groups (P<0.05) Compared with normoxia group (27.4583±0.8504), it significantly decreased in H/R group (17.9683±0.5299) (P<0.05).And compared with H/R group, that in H-PostC group (23.1117±0.3676) significantly increased (P<0.05).⑤Bax and Bcl-2 gene expression correlation analysis:a negative relationship existed between the gene expression of Bax and Bcl-2 (r=-0.765,p<0.01)Conclusion:Hypoxia can lead to typeⅡalveolar epithelial cell apoptosis via mitochondrial apoptotic pathway. Hypoxic postconditioning can obviously reduce the cell apoptosis that was induced by hypoxia.