ABSTRACT
Objective To explore the effects of endoplasmic reticulum (ER) stress in the hyperoxia-induced lung injury in premature rats. Methods Forty-eight premature Wistar rats were randomized into two groups 12 hours after birth:hyperoxia group (n=24) inhaled 95%oxygen and control group (n=24) inhaled air. Eight rats were sacriifced in each group on day 1, 3, 7 after the treatment and the left lungs were embedded. The pathological changes in the HE stained sections of lung tissues were observed. The expressions of ER related protein ERp57 and c/EBP homologous protein CHOP were detected by immuno histo-chemistry and the apoptosis of lung cells was detected by TUNEL analysis. Results The typical pathological characteristics of acute lung injury were observed in hyperoxia group. The expressions of ERp57 and CHOP were increased with the exposure time in hyperoxia group, and were signiifcantly higher than in control group (P<0.05). The apoptosis rate of lung cells in hyperoxia group was signiifcantly higher than in control group (P<0.01). There was signiifcant positive correlation between cell apoptosis index and expressions of Erp57 and c/EBP homogeneous protein. Conclusions ER stress initiated apoptosis participates and plays an important role in the process of hyperoxia-induced lung injury in premature rats.
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.
ABSTRACT
Objectives To explore the role of PKCβ/p66Shc oxidative stress signaling pathway in hyperoxia-induced apoptosis of alveolar epithelial cells A549. Methods A549 cells were cultured in vitro and divided randomly into control (incubated with 5%CO2), hyperoxia group (exposed to a mixture of 900 ml/L O2 and 50 ml/L CO2 at speed of 3 L/min for 10 mins, then cultured in a closed environment) and LY333531 group (treated with 10μmol/L of PKCβinhibitor LY333531 for 24h then induced with hyperoxia for 10 mins). The cellular morphology was observed under inverted microscope at 12, 24 and 48 h of treatment. The cell apoptosis was detected by lfow cytometry. Expression of PKCβ/Pin1/p66Shc/p66Shc-Ser36 were detected by immunohistochemistry after 24 h of treatment. Results Comparing to the control group, the cellular morphology of A549 in the hyperoxia group changed to spherical shapes and space between cells increased, the living cell count decreased and suspension cell increased. The living cell count in LY333531 group increased and suspension cell decreased than those in hyperoxia group but not reach the levels of the control group. The apoptosis rate of A549 cells and the expression of PKCβ/Pin1/p66Shc/p66Shc-Ser36 at 24 h were signiifcantly increased in the hyperoxia group than those in the control group, while the apoptosis rate and the expression of PKCβ/Pin1/p66Shc/p66Shc-Ser36 were greatly decreased in the LY333531 group than those in the hyperoxia group (all P<0.01). Conclusions The expression of PKCβin A549 cells can be increased by the hyper-oxia induction but reduced by LY333531, and then the expressions of Pin1, p66Shc and p66Shc-Ser36 are reduced. Thus the re-duced apoptosis of A549 cells relieve the cell injury induced by hyperoxia.