Research On The Roll Of Multidrug Resistant Associated Protein 4 In Vascular Endothelial Dysfunction In Sepsis

Objective:To investigate the expression of MRP4 and its regulation in the model of CLP in vivo and endotoxin induced(rat pulmonary microvascular endothelial cells)RPMVECs in vitro.Methods:1.the rat sepsis model was constructed by CLP,and the lung tissues of rats were taken at 0 h,6 h,.12 h and 24 h after CLP,respectively.The expression of MRP4 protein in lung tissue was assessed by Western blotting.2.the model of cells,gather the cells at 0 hour,6 hours,12 hours and 24 hours respectively after LPS was stimulated whose last concentration 10 ?g/ml.The expression of MRP4 protein in RPMVECs were assessed by Western blotting.Results:1.The level of MRP4 in lung were ascending regularly at 6 hours ad 12 hours,and then descending sharply.2.the level of MRP4 in RPMVECs induced by LPS were ascending regularly at 6 hours and 12 hours,and then descending sharply.Conclusion:sepsis or the stimulation of LPS affect the expression of MRP4 in lung tissues and RMPVECs.The expression of MRP4 was ascending than descending regulary.Part 2 Effects of MRP4 gene on lipopolysaccharide-induced endothelial permeability and the cytoskeleton of endothelial cells 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:Three to six passages of RPMVECs were cultured in vitro and randomly divided into four groups:control group,LPS group,Ad-shMRP4 group,Ad-shRNA group.Fluorescence microscopy was used to detect the infection efficiency of RPMVECs.The expression level of Western botting detection of MRP4.The endothelial permeability was assayed by the transwell chamber models.The morphological characteristic and distribution of F-actin 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(P<0.05),the remodeling of F-actin,and the formation of stress fibers 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 cells(P<0.05),and the remodeling of F-actin,and the formation of stress fibers in Ad-shMRP4 group..Conclusion:Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,which may play an important role in the protection of endothelial cell barrier.Part 3 Protective effect of multidrug resistant associated protein 4 inhibitor on sepsis-induced acute lung injuryObjective:To investigate the protective effect of multidrug resistant associated protein 4(MRP4)inhibitor on sepsis-induced acute lung injury in rats.Methods:Sixty Sprague-Dawley(SD)rats were randomly divided into 3 groups:control group,sepsis group and MK571 treatment group.Sepsis was induced by cecal ligation and puncture operation.Rats in MK571 treatment group were intraperitoneally injected with MRP4 inhibitor MK571(20mg/kg)30 minutes prior to operationmodel establishment.12 hours later,arterial blood PH,Pa02 and PaC02 were measured.Serum tumor necrosis-a(TNF-a)level was detected by enzyme-linked immunosorbent assay.The wet/dry weight ratio(W/D)of the lung was calculated.The expression of MRP4 protein in lung tissue was assessed by Western blotting.Results:Compared with the control group,arterial blood pH and Pa02 were significantly lower,while PaC02 was dramatically higher in the sepsis group.Serum TNF-a level in the sepsis group was significantly increased.In addition,the W/D ratio of lung tissues was significantly increased,and the expression of MRP4 protein was up-regulated in the sepsis group.Compared with the sepsis group,arterial blood pH and Pa02 were significantly elevated,while PaC02 was dramatically decreased in the MK571 treatment group.Moreover,rats in the MK571 treatment group had a lower serum level of TNF-a,reduced W/D ratio of lung tissues and down-regulated MRP4 protein expression compared with rats in the sepsis group.Conclusion:MRP4 inhibitor exerts protective effect on sepsis-induced acute lung injury in rats.