RhoA/mDia1 pathway involved in the expression of p-ERM in the pulmonary micro-vascular endothelial cell induced by lipopolysaccharide

Abstract

Objective To investigate the possibility of the involvement of RhoA/mDia1 pathway to cause the expression of phosphorylate ezrin-radixin-moesin (p-ERM) in rat pulmonary micro-vascular endothelial cell (PMVEC) after the stimulation of lipopolysaccharide (LPS). Methods The specimens of lung tissue were taken from healthy male SPF grade SD rat with 100-120 g body weight which was purchased from the laboratory animal center of Anhui province. After culture, the PMVECs were randomly divided into dose-dependent groups (0, 0.1, 1, 10 μg/mL LPS added in PMVECs and cultured for 30 min, n=8 in each), time-dependent groups (10 μg/mL LPS added to PMVECs cultured for 0, 15, 30, 60, 120 min, n=8 in each) and intervention group (n=8). In the intervention group, PMVECs were cultured with 1 μg/mL C3 transferase in serum free media for 240 min, followed by treatment with 10 μg/mL LPS for 30 min. Meanwhile, two control groups in serum-free DMEM medium were made by adding 10 μg/mL LPS to PMVECs and 1 μg/mL C3 transferase to PMVECs respectively cultured for 30 min (n=8 in each). Western blot was used to detect the level of p-ERM, ERM and mDia1. Data were analyzed with SPSS 16.0 software, while one way analysis of variance (ANOVA) was used to compare multiple sets of variables, the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests, with P<0.05 for the statistically significant difference. Results ERM, p-ERM and mDia1 were presented in rat PMVEC. Stimulation with LPS up-regulated p-ERM, mDia1 in a dose-dependent manner: LPS [0 μg/mL LPS group: (0.520±0.101), 0.1 μg/mL LPS group: (0.657±0.092), 1 μg/mL LPS group: (0.891±0.167), 10 μg/mL LPS group: (1.227±0.106); 0 μg/mL group vs. 0.1 μg/mL group, P>0.05; the rest P 0.05; the rest P 0.05; the rest P<0.05); and the level of mDia1 increased at 15 min(0.779±0.035), peaked at 30 min(0.889±0.036)then decreased at 60 min (0.648±0.019), 90 min (0.582±0.068), but kept at higher level at 120 min (0.526±0.059) than that in control group(0.284±0.118, all P<0.01). C3 transferase caused a marked attenuation of LPS induced p-ERM expression [control group: (0.339±0.069); C3+ LPS group: (0.438±0.07); C3 control group: (0.352±0.071); LPS control group: (0.634±0.191), C3+ LPS group vs. LPS control group, P=0.01], as the same in mDia1[control group: (0.449±0.122); C3+ LPS group: (0.380±0.148); C3 control group: (0.404±0.164); LPS control group: (0.622±0.174), C3+ LPS group vs. LPS control group, P<0.01]. Conclusions LPS could up-regulated the expression of p-ERM protein, and inhibition of RhoA/mDia1 signal pathway by C3 transferase could down-regulated the p-ERM levels. Key words: Lipopolysaccharide; Pulmonary microvascular endothelial cell; Ezrin-Radixin-Moesin; Inflammation; Acute respiratory distress syndrome; RhoA/mDia1