Protective Effect Of NAC In The Effects Of Oxidative Stress On The Expression Of KGFR Phosphorylation And Ubiquitinationin In Embryonic Type â…¡ Alveolar Epithelial Cells Of Rat

Objective To explore the influence of ROS to the tyrosine kinase (RTKs) and ubiquitination of KGFR in fetal rat alveolarⅡ type epithelial cells. Methods The lung tissue of19d fetal rats digested with trypsin and collagenase were isolated and purified in different centrifugal force and different adherences,then the primary fetal rat’s AECⅡ were cultured in vitro. Cells grew to subconfluence and then were randomly divided into H2O2group,NAC+H2O2group and KGF group. The H2O2group was given serum free medium containing final concentration200μM H2O2, NAC+H2O2group was first cultured for20min with serum free medium containing final concentration lOmM NAC, then was added H2O2and controled the concentration to200μM. Both of the groups were cultured in culture chamber.After cells were cultured for Omin,5min,40min,80min, the level of ROS were detected by the flow cytometry instrument.Then we detected the expression of KGFR of H2O2group in Omin,5min,10min,20min,40min and80min by western-blot method.The level of AECII KGFR phosphorylation and ubiquitination in all three groups were detected by the immune-precipitation and western-blot method. Results1.5min,40min,80min, compared with0min (air control group), the ROS level in H2O2group increased significantly (P<0.01);NAC+H2O2group compared with H2O2group, the ROS levels decreased significantly (P<0.01); but compared with0min (air control group), the level of ROS in5min and40minNAC+H2O2group is still higher (P<0.05), the level of ROS in80min NAC+H2O2group was roughly the same with0min(P>0.05).2. The level of KGFR expression in H2O2group stays the same at each time points (F test, P>0.05).3.Compared with0min(0.9379±0.0589), the ROS that produced from H2O2group in5min,10min,20min,40min activated KGFR tyrosine kinase and made KGFR phosphorylation. The peak level of KGFR phosphorylation is on5min point (1.8796±0.0973, P=0.000) and it was gradually disappear after40min (1.2775±0.0848, P=0.005), but until disappearing completely it was at80min(0.6519±0.0086).4.The KGFR tyrosine phosphorylation trend in NAC+H2O2group is consistent with H2O2group,but phosphorylation level obviously decreased.5.The ROS from H2O2group in5min,10min,20min,40min induced KGFR to ubiquitin.The peak level of ubiquitination of KGFR was at5min (2.0225±0.0402, P=0.000).It was gradually disappear from10min(1.3691±0.01386, P=0.006) to40min(1.1642±0.0032, P=0.03),but there is still a statistical significance (P<0.05) compared with0min (0.9640±0.0620).6.1t is no statistical difference that the level of KGFR phosphorylation and ubiquitination depended on KGF or ROS from5min H2O2group(P>0.05).Conclusions The activation of tyrosine kinase is mediated by Oxidative stress (ROS) production, which increased the level of KGFR phosphorylation. Sequentially,it will inhibit KGF on the protective effect of hyperoxia-induced lung injury.