| ObjectiveEstablishing cecal ligation and punture(CLP) model, to explore the effect of agmatine on the vascular endothelia biomarker as well as hepatic and pulmonary wascular permeability in sepsis mice. On this basis further investigating the secration and permeablitity of human umbilical vein endothelial cell that is activated by LPS.Preliminaryly discussing the influence of agmatine on the signal paths on HUVECs,to reveal the protective effect of agmatine on vascular endothelial dysunction and correlative mechansim,which may provide new therapeutic schedule of sepsis.MethodsSepsis model is conducted by cecal ligation and punture(CLP). 60 8-old male C57BL/6 mice were randomly divided into three groups:sham operation group(n=20),sepsis model is conducted by cecal ligation and puncture(CLP);sham operation group received laparotomy but not cecal ligation and punctureand intraperitoneally injected with phosphate buffer saline. Model group(n=20), agmatine group(n=20), mice in agmatine group intraperitoneally injected with agmatine at 400mg/kg;other two groups both intraperitoneally injected with phosphate buffer saline.Mice were sacrificed at 24 hours post-operation,and blood samples were collected for detecting the contents ofsoluble intercellular adhesion molecule-1(ICAM-1),soluble vascular cell adhesion molecule-1(VCAM-1),vascular endothelial growth factor(VEGF),von Willebrand factor(vWF),angiopoietin-2(Ang-2) and monocyte chemotactic protein-1( MCP-1), tumor necrosis factor-a(TNF-α),interleukin(IL-6 and IL-10)by enzyme linked immunosorbent assay(ELISA). Pulmonary and hepatic vascular permeability was measured using fluorescein isothiocyanate(FITC)-dextran in vivo. Hepatic tissue were collected for the observation of pathological changes by hematoxylin eosin stain.Culturing human umbilical vein endothelial cells and randomly divided into threegroups:control group, LPS group, agmatine group. The half inhibitory rate of HUVECs treated with different concentration of LPS and AGM was detected by MTT assay; The effect of agmatine on the factors that are expressed of endothelial cell were detected by enzyme linked immunosorbent assay(ELISA).24-well transwell inserts inoculated1×105cells/well, when cells formed into monolayer cell layer using endotoxin to stimulate HUVECs. After 24 h,(FITC)labeled-glucan was added,the fluorescence intensity of the upper and lower on transwell chambers were detected by fluorescence enzyme standard instrument afer 1h.LPS and AGM respectively stimulate HUVECs24 h, the extraction of total protein,via western bolt dectcting the content of ICAM-1,enothelin 1(ET-1);and agmatine preteated human umbilical vein endothelial cells 1h,then LPS stimultated endothelial cells 15,30,45,60 min,extracting the cytoplasm and cell nucleoprotein and the effect of agmatine on endothelial cell NF-κB signaling pathway was measured by westen bolt.Results(1) The role of agmatine on serum or plasma of vascular endothelial related factorsCompared with sham group, levels of vascular endothelial-related biomarkers ICAM-1, VCAM-1, VEGF, vWF, Ang-2, MCP-1 of the serum and plasma in model group was significantly increased(P <0.01), agmatine therapy could inhibit its content(P <0.01). At the same time, compared with sham, serum the levels of inflammatory cytokines IL-6, IL-10, TNF-αof serum in model group were significantly increased(P<0.01), while the expression of inflammatory mediators after agmatine treatment was significantly decreased(P < 0.01).(2) Effect of agmatine on septic liver,lung wascular permeabilityThe FITC-dextran in the lung and liver lysates from CLP model was significantly increased when compared with sham group, and AGM treatment could reverse the increase. Hepatic tissue histological significantly changed after CLP, assuming hepatocytes swelling,hyperaemia and neutrophils infiltration, while AGM obviouslyreduced liver injury.(3) The effect of agmatine on endotoxin induced human umbilical vein endothelialcellsThe content of ICAM-1,IL-6 increased significantly after LPS stimulatedHUVECs, while the secretion of above factors of endothelial cells were significantreduced when agamtine was added. the fluorescence intensity of lower transwellchamber was obviously higher, after AGM treatment the fluorescence intensity wassignificantly reduced(P﹤0.05)(4) Effect of agmatine on protein and signaling molecules of HUVECs after LPSstimulationIn LPS group, the ICAM-1,ET-1 protein expression of endothelial cells wereoutstandtingly higher,agmatine group the protein of ICAM-1 and ET-1 were obviouslylower than LPS group. LPS15, 30 min group endothelial cells protein p65, p50 weresignificantly higher, agmatine 15,30 min group nucleoprotein p65, p50 was remarkablylower than LPS group.ConclusionAgmatine can ameliorate vascular endothelial dysfunction of septic mice via inhibiting the expression of inflammatory factors and vascular endothelia biomarkers and decreasing hepatic and pulmonary vascular permeability. Agmatine could improve human umbilical vein endothelial cell function and permeability, its effect may be mediated through the NF-κB signaling pathway... |
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