Protective Effects Of BML-111,a Lipoxin Receptor Agonist, On Sepsis-induced Pulmonary Endothelial Hyperpermeability In Mice

Part I The protective effects of lipoxin receptor agonist BML-111on sepsis-induced acute lung injury in miceObjective To investigate the protective effect of lipoxin receptor agonist BML-111on sepsis-induced acute lung injury in mice.Method SPF grade BALB/c mice, approximately25g in weight, were randomly divided into the following five groups:the blank group (Group Control), the sham group (Group BML-111), the sepsis group (Group LPS), the treatment group (Group BML-111+LPS) and the antagonist group (Group Boc-2). The Group LPS received intraperitoneal injection of lipopolysaccharide (lOmg/kg); the Group Control received intraperitoneal injection of normal saline; the Group BML-111+LPS and the group Boc-2received intraperitoneal injection of BML-111(lmg/kg), followed by intraperitoneal injection of lipopolysaccharide (10mg/kg) after30minutes; while the Group BML-111only received intraperitoneal injection of BML-111(lmg/kg); the Group Boc-2received intraperitoneal injection of Boc-2(50μg/kg)15min before intraperitoneal injection of BML-111. The survival rate and the body weight loss of the mice were recorded..All of the5groups of mice were sacrificed and the lungs were lavaged24hours after the stabilizing the model. Under light microscope, histological changes were scored according to the lung injury. The leukocyte counts in bronchoalveolar lavage fluid (BALF) were determined. The level of tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6) in bronchoalveolar lavage fluid(BALF) and lung tissue were both assayed by ELISA.Results Compared with the Group Control, the mortality rate of Group LPS increased obviously, and the weight reduced significantly at the time point of24hours after modeling(p<0.01).; the alveolar structure obviously changed in the Group LPS with a significantly higher lung injury score(p<0.01; the leukocyte count in the BALF, the concentration of TNF-α and IL-6in the BALF and lung tissue significantly increased(p<0.01). Compared with the Group LPS, the Group BML-111+LPS showed a obvious improvement of the survival rate of mice and weight change significantly reduced(p<0.01); BML-111significantly reduced the leukocyte counts in the BALF and inhibited the expression of IL-6and TNF-alpha in lung and bronchoalveolar lavage fluid (BALF)(p<0.01). Boc-2can block the inhibitory effect of BML-111in the inflammatory response(p<0.01).Conclusion BML-111can improve the symptoms of sepsis in mice and reduce the degree of the pathological changes of sepsis-induced acute lung injury with reduction of neutrophil infiltration and pro-inflammatory cytokines expression in lung tissue. The lipoxin receptor agonist BML-111had a protective effect on sepsis-induced acute lung injury in mice. Part II The effect of lipoxin receptor agonist BML-111on lung microvascular permeability with sepsis in miceObjective To explore the effect of lipoxin receptor agonist BML-111on lung microvascular permeability with sepsis in mice and the expression of the lung endothelial adherence junction protein.Method SPF grade BALB/c mouse, approximately25g in weight, were randomly divided into the following five groups:the blank group (Group Control),the sham group (Group BML-111), the sepsis group (Group LPS), the treatment group (Group BML-111+LPS) and the antagonist group (Group Boc-2). The Group LPS received intraperitoneal injection of lipopolysaccharide (1Omg/kg); the Group Control received intraperitoneal injection of normal saline; the Group BML-111+LPS and the Group Boc-2received intraperitoneal injection of BML-111(lmg/kg), followed by intraperitoneal injection of lipopolysaccharide (10mg/kg) after30minutes; while the Group BML-111only received intraperitoneal injection of BML-111(lmg/kg); the Group Boc-2received intraperitoneal injection of Boc-2(50μg/kg)15min before intraperitoneal injection of BML-111. All of the5groups of mice were sacrificed and the lungs were lavaged24hours after the stabilizing the model. The wet to dry ratio (W/D) of lung tissues was assayed. The total protein concentration in the bronchoalveolar lavage fluid (BALF) were measured. The permeability of lung endothelium was detected using Evans Blue method. The expressions of lung endothelial adherence junction proteins were tested using Western Blot.Results Compared with the Group Control, in the Group LPS, the wet to dry ratio (W/D) of lung tissues, the total protein concentration in the BALF and the exudation of Evans Blue in the lung tissue increased significantly(p<0.01). The expression of ZO-1and VE-Cadherin of lung tissue in the Group LPS decreased significantly (p<0.05). While compared with the group LPS, BML-111can significantly reduce the lung tissue edema (W/D ratio)(p<0.01), inhibit lung microvascular permeability of Blue Evans (p<0.01), reduce the protein concentration in bronchoalveolar lavage fluid (BALF)(p<0.01), and up-regulate the protein expression level of ZO-1and VE-Cadherin (p<0.05) in the Group BML-111+LPS.Conclusion Lipoxin receptor agonist BML-111can obviously reduce pulmonary edema and exudation induced by pulmonary vascular endothelial permeability. BML-111can maintain pulmonary vascular endothelial integrity and alveolar-capillary barrier function through the molecular mechanisms of regulation in ZO-1and VE-Cadherin. Part III The effect of lipoxin receptor agonist BML-111on adherence junction proteins expression on endothelial cellObjective To explore the effect of lipoxin receptor agonist BML-111on adherence junction proteins expression such as VE-Cadherin and ZO-1in lipopolysaccharide(LPS)-stimulated human endothelial cell.Methods Human endothelial cell EA.hy926were cultured in vitro and were divided into five groups:the blank group (Group Control), the control group (Group BML-111), the stimulated group (Group LPS), the treatment group (Group BML-111+LPS) and the antagonist group (Group Boc-2). The Group LPS were treated with1μg/ml lipopolysaccharide under serum-free conditions; the Group Control were treated with serum-free conditions; the Group BML-111+LPS were treated with BML-111(50nM,200nM) and the Group Boc-2were treated with BML-111200nM, followed by addition of lμg/ml lipopolysaccharide after30minutes; while the Group BML-111were only treated with BML-111200nM; the group Boc-2were treated with100μM Boc-2prior to the treatment with BML-111for15min.The expression of VE-Cadherin and ZO-1on endothelial were detected by Western blot and immunofluorescence. The effect of BML-111on LPS-induced phosphorylation of Akt and MAPKs were detected by Western blot.Results Western blot and immunofluorescence both showed that the expression of ZO-1and VE-Cadherin were inhibited in the1μg/m LPS treated endothelial cell. In the Group BML-111+LPS, with the pre-treatment of200nM BML-111, the expression of ZO-1and VE-Cadherin were up-regulated and the endothelial cells returned to normal.200nM BML-111could also inhibit the phosphorylation of Akt and MAPKs in the LPS stimulated endothelial cells.Conclusion Lipoxin receptor agonist BML-111could up-regulate the expression of adherence junction proteins expression such as VE-Cadherin and ZO-1in lipopolysaccharide(LPS)-stimulated human endothelial cell, which leads to the maintenance of the endothelial cell skeleton structure and endothelial barrier function.