[Role of Ezrin in the injury of rat pulmonary microvascular endothelial cells induced by tumor necrosis factor-α and the impact of Rac 1]

Abstract

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-α and intervention group in which cells were pretreated with Rac 1 inhibitor (NSC 23766). (1) In the time-dependent injury group, Western Blot was used to detect the expression of Ezrin and p-Ezrin after 10 μg/L TNF-α stimulation for 0, 0.25, 0.5, 1, 3, 6, 12, 24 hours. (2) In the intervention group, after pre-treatment with 200 μmol/L NSC 23766 for 0.5 h, PMVECs were treated with 10 μg/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-α stimulation 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-α could increase Ezrin phosphorylation in a time-dependent manner. (2) Compared with blank control group, in single NSC 23766 or TNF-α simulation group, p-Ezrin expression was induced (TNF-α group: 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-α simulation 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).

Conclusions: 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.