Expression of NF-κB and Bak in oxidative stress-induced apoptosis of alveolar type Ⅱ epithelial cells

Abstract:

Objective：To investigate the changes of nuclear factor-κB（NF-κB） p65 and Bak expression during the apoptosis of type Ⅱ（ATⅡ） epithelial cells induced by oxidative stress，and the apoptotic outcomes of ATⅡ cells as well. Methods：Primary ATⅡ cells were separated，purified and cultured from adult Sprague-Dawley rats treated with 500 μmol/L hydrogen peroxide（H2O2） for different periods of time，with the purpose of establishing an apoptotic cell model by oxidative injuries. The change of cell morphology was ex－plored by phase contrast microscope. Apoptotic ratio was measured by flow cytometry. The changes in expression of NF-κB p65 and Bak were investigated by Western blot. The location of NF-κB p65 was detected by immunofluorescence and observed under laser con－focal microscopy. Results：After exposure to H2O2 for 3 h，ATⅡ cells turned smaller，with karyopycnosis and reduction of cytoplasmic granules. The cell apoptotic ratios after exposure to H2O2 for 1 h and 3 h were both increased obviously（P=0.000，P=0.000），compared with those of normal control group. Western blot detected that the nucleic expression of NF-κB p65 and the expression of Bak after exposure to H2O2 for 3 h were increased remarkably compared with those of normal control group（P=0.000，P=0.000）. The nucleic translocation of NF-κB p65 after H2O2 treatment was also increased in the merged images under laser confocal microscopy. Conclusion：High level of oxidative stress induces ATⅡcells apoptosis and up-regulates the nuclei expression of NF-κB p65 and the expression of Bak.