Role of adapter protein p66Shc in injured mediating alveolar epithelial cells induced by hyperoxia

Abstract

Objective To determine the expression of adapter protein p66Shc in mediating alveolar epithelial cells induced by hyperoxia and to explore their relationship. Methods A549 cells were cultured in vitro and divided randomly into a control group and a hyperoxia group.The hyperoxia group was exposed to a mixture of oxygen(O2, 900 mL /L) and carbon dioxide(CO2, 50 mL /L) for 10 min, then cultured in a closed environment.The changes in morphology were observed under inverted microscope after exposure to oxygen or air for 24 hours.The cell apoptosis was detected by flow cytometry(FC) after 24 hours.And the expression of p66Shc was detected by immunohistochemical method after 24 hours.The correlation of the changes in mitochondrial membrane potential and p66Shc protein expression was analyzed by using Bivariate correlation analysis. Results 1.Under inverted microscopy, A549 cells from the air group significantly increased, stuck to each other tightly and grew very quickly.Their adhesion was better, multy-angle oblate and many cells were in division phase.Compared with the control group, the changes in morphology of A549 were remarked and obvious than those in the hyperoxia group.The cells grew slowly, their counts decreased and the cell morphology changed from typical multi-angle oblate to round or ellipse.2.Compared with the control group, after 24 h, in hyperoxia group of A549 cells, red fluorescence decreased, and green fluorescence enhanced.3.Compared with the controls(0.057 664 88±0.006 517 84), the expression of p66Shc(0.123 600 50±0.004 227 23) was significantly increased in the hyperoxia group(t=-24.006, P<0.001).4.The decline of membrane potential was negatively correlated with the increased expression of p66Shc protein(R=-0.988, P<0.001). Conclusions The hyperoxia induction could significantly increase in injured mediating alveolar epithelial cells induced by hyperoxia, the expression of p66Shc increases, the membrane potential declined, and they exhibit a negative correlation.So p66Shc may be involved in the process of high oxygen damage to human alveolar epithelial cells. Key words: Hyperoxia; Alveolar epithelial cells; Adapter protein; Oxidative stress; Apoptosis