RESUMO
Objective 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.Methods PMVECs of rats were culturedin vitroand 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 10μg/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, 10μg/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 (200μmol/L) for 0.5 hour or Rac1 specific agonist O-Me-cAMP (200μmol/L) for 1 hour, respectively, and then incubated with 10μg/L TNF-α for 6 hours. The PMVECs without treatment were served as blank control group, andthose 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.Results ① Time-effect experiment results: the expression of Moesin showed no change among all time points after 10μg/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 0.1μg/L as compared with0μg/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 1μg/L and 10μg/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).Conclusion 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.
RESUMO
Objective To investigate the role of Ezrin and its phosphorylation(p-Ezrin)in the modulation of rat pulmonary microvascular endothelial cell(PMVEC)injury induced by tumor necrosis factor-α(TNF-α)and the impact of Rac 1. Methods Cultured PMVECs of Sprague-Dawley(SD)rats were randomly divided into time-dependent injury group induced by TNF-αand intervention group in which cells were pretreated with Rac 1 inhibitor (NSC 23766).①In the time-dependent injury group, Western Blot was used to detect the expression of Ezrin and p-Ezrin after 10μg/L TNF-αstimulation for 0,0.25,0.5,1,3,6,12,24 hours.②In the intervention group,after pre-treatment with 200μmol/L NSC 23766 for 0.5 h,PMVECs were treated with 10μg/L TNF-α,and the expression of p-Ezrin was detected by Western Blot after 3 hours. Besides these groups,there were control(1% fetal bovine serum simulation),single NSC 23766 or TNF-α simulation groups. Results ① Few Ezrin expression was found in PMVEC,and TNF-α could not affect Ezrin expression. p-Ezrin protein expression(p-Ezrin/Ezrin,gray scale) of PMVECs at 0 hour after TNF-αstimulation was 0.21±0.03,and elevated at 0.25 hour(0.53±0.19),peaked at 3 hours(1.68±0.30),then it was gradually lowered,but it remained at higher level at 24 hours(0.87±0.18)with significant difference(F=62.200,P=0.000). It demonstrated that TNF-αcould increase Ezrin phosphorylation in a time-dependent manner.②Compared with blank control group,in single NSC 23766 or TNF-αsimulation group, p-Ezrin expression was induced(TNF-αgroup:0.92±0.12 vs. 0.68±0.16,t=-2.864,P=0.020;NSC 23766 group:1.33±0.24 vs. 0.68±0.16,t=-5.429,P=0.000. When NSC 23766 was pre-treated with PMVECs,the expression of p-Ezrin was significantly increased compared with that in single TNF-αsimulation group(2.14±0.18 vs. 0.92±0.12,t=-14.670,P=0.000)with significant difference(F=73.810,P=0.000). Conclusion Ezrin proteins are phosphorylated by TNF-α. Rac 1 signaling pathway inhibition plays an important role in TNF-α-induced injury by up-regulation of p-Ezrin in PMVECs.