Effects of MRP4 gene on lipopolysaccharide-induced endothelial permeability and the cytoskeleton of endothelial cells changes / 中华急诊医学杂志

ABSTRACT

Objective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groupscontrol group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.