Role Of Phosporylated Moesin In The Injury Of Pulmonary Microvascular Endothelial Cells Of Rats And Its Mechanism

Objective: To investigate the role of phosphorylated Moesin(p-Moesin)in the injury of pulmonary microvascular endothelia cells(PMVECs)of rats induced by tumor necrosis factor-α(TNF-α),and to approach the impact of Rac1 signal pathway on Moesin phosphorylation.Motheds: PMVECs of rats were cultured in vitro and passed on to the third generation,and,the TNF-α time-effect experiment,dose-effect experiment and Rac1 signaling pathway intervention experiment were performed respectively.In time-effect experiment,PMVECs were stimulated with 10μg/L TNF-α for 0,15,30 minutes and 1,3,6,12 hours.In dose-effect experiment,PMVECs were stimulated with 0,0.1,1,10 μg/L TNF-α for 6 hours.In intervention experiment,PMVECs were divided into two parts,which were pretreated with 3 mL Rac1 specific inhibitor NSC23766(200μmol/L)for 0.5h Rac1 specific agonist O-Me-cAMP(200μmol/L)for 1 hour,respectively,and then incubated with 10μg/L TNF-α for 6 hours.All of those were compared with blank control groups.The protein expression of Moesin and p-Moesin were determined by Western-blot.Result: 1.The rat PMVECs was isolated and cultured in vitro successfully.Through the morphology and the combining experiment with Fluorescein isothiocyanate(FITC)labeled Plant lectin,we confirmed that the cell was rat PMVECs.2.The expression of Moesin protein in rat PMVECs was detected in normal control group by Western-blot method,and p-Moesin protein was expressed on a low level.3.The expression of Moesin showed no change among all time points after 10μg/L TNF-α stimulated PMVECs.But the expression of p-Moesin protein was sharply up-regulated at 15 minutes,peaked at 30 minutes,and the gradually decreased after 1 hour,but still higher at 12 hour then 0 hour group.It indicated that TNF-α up-regulated the expression of Moesin protein in a time-dependent manner.No significant change in expression of Moesin protein was found among all dose of TNF-α incubated with rat PMVECs for 6 hours.But the expression of p-Moesin protein was showed an upward trend with 0、 0.1、1、10ug/L TNF-α stimulation.It indicated that TNF-α up-regulated the expression of Moesin protein in a does-dependent manner.4.Compared with blank control group,Rac1 specific agonist O-Me-cAMP or Rac1 specific inhibitor NSC23766 alone could not change the expression of p-Moesin Protein.Compared with TNF-α group,the expression of p-Moesin protein induced by TNF-α was up-regulated by NSC23766,and it was attenuated by O-Me-cAMP.Conclusion: 1.We isolated and cultured rat PMVECs successfully,also confirmed that Was rat PMVECs.2.The expression of Moesin and p-Moesin were detected in rat PMVECs.3.The expression of Moesin was not changed by TNF-α,but the expression of p-Moesin in PMVECs of rats was increased by TNF-α in a time and concentration dependent manner.4.Activation of Rac1 signaling pathway can significantly reduce the expression of p-Mosein protein in rat PMVECs induced by TNF-α.Inhibition of Rac1 signaling pathway significantly up-regulated the expression of p-Mosein protein in rat PMVECs induced by TNF-α.