The Role Of PKCβ/p66Shc Oxidative Stress Signal Pathway In Mediating Alveolar Epithelial Cells Apoptosis Induced By Hyperoxia

Objective:To explore the role of PKCβ/p66Shc oxidativestress signal pathway in mediating apoptosis of hyperoxia-exposed alveolar epithelial cells. To explore the role of PKCβ inhibitor LY333531 protective effect in alveolar epithelial cells induced by hyperoxia.Methods:This study is based on alveolar type Ⅱ epithelial cell line A549 cells (A549) in vitro subculture as the research object. Human alveolar epithelial A549 cells (A549) were cultured in vitro and divided randomly into control, hyperoxia group and LY333531 group. The hyperoxia group was exposed to a mixture of O2(950ml/L)and CO2(50ml/L) for 10 minutes, then cultured in a closed environment. The LY333531 group was treated with specific PKCp inhibitor LY333531 of 10μmol/L for 24h before hyperoxia induction. Cells were collected, and then, the change of morphology were observed under inverted microscope after exposure to oxygen or room air for 12,24 and 48 hours; the expression of protein PKCβ/Pinl/p66Shc/p66Shc-ser36 were detected by immunohistochemical methods after 24 hours; also, the expression of protein PKCβ/Pinl/p66Shc/p66Shc-ser36 were detected by Western blotting procedures after 24 hours; the detection of intracellular translocation about p66Shc, the production of ROS and cellular mitochondria membrane potential (ΔΨm) was used by confocal microscopy; The cell apoptosis was detected by flow cytometry (FC) after 24 hours.Results:(1)Under the inverted microscopy, in air group, A549 cells growth in good condition, were significantly increased, grew quickly and sticked to each other tightly, their adhesion were better, multy-angle oblate, many cells were in division phase. Compared with control group, the changes in morphology of A549 were most obvious in the hyperoxia group, the cells grew slowly, the mount decreased, form of the cells changed from typical multy-angle oblate to ellipse or round. Gap between cells were increased, and contour enhanced. How ever, the changes in morphology of A549 were obviously decreased in LY333531 group, but not reached the control group. (2) Immunohistochemical results showed the following, compared with control group, the expression of PKCβ/Pin1/p66Shc/ p66Shc-ser36 were significantly increased in the hyperoxia group(P<0.05); But compared with the hyperoxia group, the expression of Pin1/p66Shc/p66Shc-ser36 were significantly decreased in the LY333531 group(P<0.05), but not reached the control group. (3)Western-blot results showed the following, compared with control, the expression of PKCβ/Pinl/p66Shc/p66Shc-ser36 were significantly increased in the hyperoxia group(P<0.05), but compared with the hyperoxia group, the expression of Pin1/p66Shc/p66Shc-ser36 were significantly decreased in the LY333531 group(P<0.05), but not reached the control group(P<0.05). (4) Confocal laser scanning microscopic showed the following, compared with control group, translocation of p66Shc from cytoplasm into mitochondria were increased significantly(P<0.05), compared with the hyperoxia group, the translocation of p66Shc from cytoplasm into mitochondria were decreased(P<0.05), but not reached the control group(P<0.05). (5) Confocal laser scanning microscopic showed the following, compared with control group, the production of mitochondrial ROS were increased significantly(P<0.05), but compared with the hyperoxia group, the production of mitochondrial ROS were decreased(P<0.05), but not reached the control group(P<0.05). (6) Confocal laser scanning microscopic showed the following, compared with control group, the mitochondrial membrane potential were decreased significantry(P<0.05), but compared with the hyperoxia group, the production of mitochondrial ROS were increased(P<0.05), but not reached the control group(P<0.05). (7) Flow cytometry showed the following, compared with the control group, the apoptosis rate of A549 cells was significantly increased in the hyperoxia group; But compared with the hyperoxia group, the apoptosis rate of A549 cells was obviously decreased in the LY333531 group(P<0.05), but not reached the control group(P<0.05).Conclusion:The hyperoxia induction can significantly increace the expression of PKCβ in A549 cells, and increace the expression of Pinl/p66Shc in A549 cells, and phosphorylation, and increace the expression of p66Shc-ser36 in A549 cells, and transport to mitochondrion, increace the ROS production, decrease the mitochondrial membrane potential and it increace apoptosis rate of A549 cells. On the contrary, The LY333531 could reduce the expression of PKCP, reduce apoptosis rate of A549 cells to relieve the A549 cell’s injury induced by hyperoxia. PKCβ/p66Shc oxidative stress signal pathway mediated apoptosis of hyperoxia-exposed alveolar epithelial cells.