Role of phosphorylated Moesin in the injury of pulmonary microvascular endothelial cells of rats and its mechanism

Abstract

To investigate the role of phosphorylated Moesin (p-Moesin) in the injury of pulmonary microvascular endothelial cells (PMVECs) of rats induced by tumor necrosis factor-α (TNF-α), and to approach the impact of Rac1 signal pathway on Moesin phosphorylation. 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. (1) Time-effect experiment: PMVECs were stimulated with 10 μg/L TNF-α for 0, 15, 30 minutes and 1, 3, 6, 12 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. (2) Dose-effect experiment: PMVECs were stimulated with 0, 0.1, 1, 10 μg/L TNF-α for 6 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. (3) Rac1 signaling pathway intervention experiment: PMVECs were divided into two parts, which were pretreated with 3 mL Rac1 specific inhibitor NSC23766 (200 μmol/L) for 0.5 hour or Rac1 specific agonist O-Me-cAMP (200 μmol/L) for 1 hour, respectively, and then incubated with 10 μg/L TNF-α for 6 hours. The PMVECs without treatment were served as blank control group, and those were treated with only O-Me-cAMP, NSC23766 or TNF-α were served as corresponding groups. The protein expressions of Moesin and p-Moesin were determined by Western Blot. (1) Time-effect experiment results: the expression of Moesin showed no change among all time points after 10 μg/L TNF-α stimulated PMVECs. But the expression of p-Moesin was sharply up-regulated at 15 minutes after TNF-α stimulation as compared with 0 minute (p-Moesin/Moesin: 4.399±0.523 vs. 1.000±0.195), peaked at 30 minutes (6.069±0.557), and then gradually decreased after 1 hour (5.005±0.544, 4.599±0.478, 1.742±0.288, 1.503±0.352 at 1, 3, 6, 12 hours, respectively) with significant difference among all time points (F = 15.397, P = 0.002). (2) Dose-effect experiment results: no significant change in expression of Moesin was found among all doses of TNF-α incubated with PMVECs for 6 hours. But the expression of p-Moesin was significantly up-regulated after TNF-α stimulation with 0.1 μg/L as compared with 0 μg/L (p-Moesin/Moesin: 2.194±0.430 vs. 1.000±0.273), which showed an upward trend with dose increase in TNF-α (3.201±0.688 and 4.413±0.296 with 1 μg/L and 10 μg/L TNF-α, respectively) with significant difference among all doses (F = 92.513, P < 0.001). (3) Rac1 signaling pathway intervention experiment results: there was no significant difference in Moesin expression among all the groups. 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, while TNF-α could induce p-Moesin expression. Compared with TNF-α group, the expression of p-Moesin induced by TNF-α was up-regulated by NSC23766 (p-Moesin/Moesin: 2.612±0.355 vs. 1.911±0.297, P < 0.05), and it was attenuated by O-Me-cAMP (p-Moesin/Moesin: 1.928±0.331 vs. 3.030±0.353, P < 0.05). The phosphorylation of Moesin is involved in the damage of TNF-α-induced PMVECs in rats, and PMVECs damage could be alleviated by modulating Moesin phosphorylation in the Rac1 signaling pathway.