An Experiment-based Study On Facilitated Removal Of Alveolar Edema Fluid By Lipoxin Agonist BML-111 In Endotoxin-Induced Acute Lung Injury

PartⅠThe protective effect of lipoxin agonist BML-111 on endotoxin-induced acute lung injury in ratsObjective To establish a model for endotoxin-induced acute lung injury in rats, and to discuss the protective effect of lipoxin agonist BML-111 on endotoxin-induced acute lung injury and the possible mechanism involved.Method 40 clean SD rats, approximately 250～350g in weight, were randomly divided into the following 4 groups:the control group (Group C), the lipopolysaccharide group (Group A), the BML-111 Group (Group B) and the BML-111 treatment group (Group AB). The Group C received intraperitoneal injection of PBS, followed by intravenous injection of lipopolysaccharide (5mg/kg) after 30 minutes; The Group B received intraperitoneal injection of BML-111 (1μg/g), followed by intravenous injection of normal saline; The Group AB received intraperitoneal injection of BML-111 (1μg/g), followed by intravenous injection of lipopolysaccharide (5mg/kg) after 30 minutes. All of the 4 groups of rats were sacrificed 6 hours after intravenous injection of normal saline and lipopolysaccharide. The pathological changes of the rats' lung tissues were observed using light microscope, and the acute lung injuries were rated; The wet to dry ratio (W/D) of lung tissues was assayed; The total protein concentration in the BALF was measured; The activity of myeloperoxidase (MPO) of the lung tissues was assayed; The concentration and of malondialdehyde (MDA) and the vitality of superoxide dismutase (SOD) both were assayed; The lung tumor necrosis factorα(TNF-α) mRNA was detected using reverse transcription- polymerase chain reaction (RT-PCR) method; The activation level of NF-κB of rat lung tissues was tested using EMSA method.Results (1) There were no significant changes of alveolar structure in the control group and the BNL-111 group, while alveolar capillary congestion, inflammatory cell infiltration and increased alveolar septum were observed in the lipopolysaccharide group and the BML-111 group. Compared with the control group, the ratings of acute lung injuries in the lipopolysaccharide group and the BML-111 group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the rating of acute lung injuries in the BML-111 treatment group decreased significantly (P<0.05). (2) Compared with the control group, the wet to dry ratios in the lipopolysaccharide group and the BML-111 group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the wet to dry ratios in the BML-111 treatment group decreased significantly (P<0.05). (3) Compared with the control group, the total protein concentration in the BALF in lipopolysaccharide group and the BML-111 treatment group increased significantly (P <0.05); while compared with the lipopolysaccharide group, the total protein concentration in the BALF in the BML-111 treatment group decreased significantly (P<0.05). (4) Compared with the control group, the activity of the MPO of lung tissue in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the activity of the MPO of lung tissue in the BML-111 treatment group decreased significantly (P<0.05). (5) Compared with the control group, the NO level in lung tissue in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the NO level in lung tissue in the BML-111 treatment group decreased significantly (P<0.05). (6) Compared with the control group, the vitality of the SOD of lung tissue in the lipopolysaecharide group and the BML-111 treatment group decreased significantly (P<0.05); while compared with the lipopolysaccharide group, the vitality of the SOD of lung tissue in the BML-111 treatment group increased significantly (P<0.05). (7) Compared with the control group, the TNF-a mRNA level in lung tissue in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the vitality of the TNF-a mRNA level in lung tissue in the BML-111 treatment group decreased significantly (P<0.05). (8) Compared with the control group, the activation level of NF-κB in lung tissue in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the activation level of NF-κB in lung tissue in the BML-111 treatment group decreased significantly (P<0.05).Conclusion The lipoxin agonist BML-111 had a protective effect on endotoxin-induced acute lung injury in rats, and the involved mechanism could be related to resistance of oxidative damage, reduction of inflammatory cell infiltration, suppression of the activation of NF-κB and the expression of TNF-a mRNA. PartⅡThe effect of lipoxin agonist BML-111 on alveolar fluid clearance and on a-ENaC in lung fluid transport with acute lung injury in ratsObjective To discuss the effect of lipoxin agonist BML-111 on alveolar fluid clearance and on a-ENaC in lung fluid transport with acute lung injury in rats.Method 40 clean SD rats, approximately 250～350g in weight, were randomly divided into the following 4 groups:the control group (Group C), the lipopolysaccharide group (Group A), the BML-111 Group (Group B) and the BML-111 treatment group (Group AB). The Group c received intraperitoneal injection of PBS, followed by intravenous injection of lipopolysaccharide (5mg/kg) after 30 minutes; The Group B received intraperitoneal injection of BML-111 (1μg/g), followed by intravenous injection of normal saline; The Group AB received intraperitoneal injection of BML-111 (1μg/g), followed by intravenous injection of lipopolysaccharide (5mg/kg) after 30 minutes. The tracheotomy was given 6 hours after all all of the 4 groups of rats got intravenous injection of normal saline and lipopolysaccharide. And the rats were connected to ventilator for small animals for volume control ventilation. The ventilation parameters are as follows:tidal volume 10 ml/kg, positive end-expiratory pressure 3cmH2O and respiratory rate 40-60times/min, the oxygen concentration 100%. The carotid artery on one side was separated, and the ductus arteriosus was used, and both pressure transducer and monitor were connected to momitor the vital signs on an on-going basis. The blood sample was collected through this way. The arterial blood gas analysis was conducted. After the circulation stabilized for 30 minutes, each indicator was measured. The changes of mean arterial pressure (MAP), heart rate (HR) and blood gas were measured; the method used to assay the alveolar fluid clearance (AFC) is as follows:the 5% bovine serum albumin labeled by FITC-albumin was injected into the lung of rats, and the rats were sacrificed after mechanical ventilation for 1 hour. Then, the entire lung, trachea and heart were removed and the alveolar fluid was aspirated. And then, a fluorescence spectrophotometer was used to read the values of labeled albumin before and after injection. The equation AFC (%)=[(Cf-Ci)/Cf]×100%was used to calculate the alveolar fluid clearance (AFC), in which, Ci denotes the value of labeled albumin before injection, Cf denotes the value of labeled albumin after aspiration. The pathological changes of lung tissue was observed and the rating of acute lung injury was assayed; The reverse transcription-polymerase chain reaction (RT-PCR) method was used to measure the level of a-ENaC mRNA expression in lung tissue. And the Western Blot analysis was used to measure the a-ENaC expression in lung tissue.Results (1) There were no significant differences in MAP, HR and blood gas between the blank control group and the BML-111 group (P>0.05); compared with the control group, the HR and PaaCO2 in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05), while the MAP, PH and PaO2 decreased significantly (P<0.05); compared with the lipopolysaccharide group, the HR and PaCO2 in the BML-111 treatment group decreased significantly (P<0.05), while the MAP, PH and PaO2 increased significantly (P<0.05); (2) Compared with the control group, the AFC calculated in the lipopolysaccharide group decreased significantly (P<0.05); Compared with control group, the AFC calculated in the BML-111 group increased significantly (P<0.05); while compared with the lipopolysaccharide group, the AFC calculated in the BML-111 treatment group increased significantly (P<0.05). (3) The lung tissue structure maintained integrity in the control group and the BML-111 group; the alveolar septum was normal; there was no inflammatory cell infiltration; while the lung tissue structure was obviously damaged in the lipopolysaccharide group, there was large amount of inflammatory cell infiltration, and the alveolar septum increased; As to the BML-111 treatment group, there was inflammatory cell infiltration, the alveolar septum increased, however, the pathological change was less than the lipopolysaccharide group. Compared with the control group, there was no significant change of lung injury rating in the BML-111 group (P<0.05), while the lung injury ratings in the lipopolysaccharide group and the BML-111 treatment group increased significantly (P<0.05); compared with the lipopolysaccharide group, the lung injury rating in the BML-111 treatment group decreased significantly (P<0.05); (4) Compared with the control group, the expression ofα-ENaC mRNA in the LPS group decreased significantly (P<0.05), the expression ofα-ENaC mRNA in the BML-111 group and BML-111 treatment group increased significantly (P<0.05); there was no significant difference in expression ofα-ENaC mRNA between the BML-111 group and BML-111 treatment group(P>0.05); (5) compared with the control group, the expression ofα-ENaC in the LPS group decreased significantly (P<0.05); the expression ofα-ENaC in the BML-111 group and the BML-111 treatment group increased significantly (P<0.05), while the expression ofα-ENaC in the BML-111 and the BML-111 treatment group increased significantly(P<0.05); there was no significant difference in expression ofα-ENaC between the BML-111 group and BML-111 treatment group(P>0.05).Conclusion The lipoxin agonist BML-111 can promote the alveolar fluid clearance in rats, thus has an clearing effect on the alveolar fluid in the case of endotoxin-induced acute lung injury, which is possibly achieved through enhancing the expression ofα-ENaC in lung fluid transport. PartⅢThe effect of lipoxin agonist BML-111 on level of lipopolysaccharide-induced cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in typeⅡalveolar epithelial cellObjective To study the effect of lipoxin agonist BML-111 on the level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in typeⅡalveolar epithelial cell (A549)Method A549 typeⅡalveolar epithelial cells were cultured in vitro and were randomly divided into the following 4 groups:①Blank control group②LPS group:using lipopolysaccharide (1μg/ml) to stimulate A549 typeⅡalveolar epithelial cells③BML-111 group:treating the A549 typeⅡalveolar epithelial cells using BML-111 (1μg/ml)④BML-111+LPS group:Pretreatment using BML-111 (1μg/ml), then using lipopolysaccharide (1μg/ml) to stimulate A549 typeⅡalveolar epithelial cells. Each group was incubated for 6 hours, then the apoptosis was measured using flow cytometry. The expression ofα-ENaC mRNA was measured using reverse transcription-polymerase chain reaction (RT-PCR). The expression ofα-ENaC was measured using Western Blot method. And the content of cAMP and cGMP in the A549 typeⅡalveolar epithelial cells was assayed using radioimmunoassay.Results (1) Compared with the control group, apoptosis in the LPS group and the BML-111+LPS group increased significantly (P<0.05); compared with the LPS group, the apoptosis in the BML-111+LPS group decreased significantly(P<0.05);(2)Compared with the control group, the expression ofα-ENaC mRNA in the LPS group decreased significantly (P<0.05), the expression ofα-ENaC mRNA in the BML-111 group and the BML-111+LPS group increased significantly (P<0.05); (3) Compared with the control group, the expression of a-ENaC in the LPS group decreased significantly (P<0.05); the expression of a-ENaC in the BML-111 group and the BML-111+LPS group increased significantly (P<0.05); (4) Compared with the control group, the level of cAMP in cells in the LPS group decreased significantly (P<0.05); the level of cAMP in cells in the BML-111 group and the BML-111+LPS group increased significantly (P<0.05). Compared with the control group, the level of cGMP in the LPS group increased significantly (P<0.05), while the level of cGMP in the BML-111 group and the BML-111+LPS group decreased significantly (P<0.05).Conclusion The lipoxin agonist BML-111 can increase the level of cyclic adenosine monophosphate (cAMP) and decrease the level of cyclic guanosine monophosphate (cGMP) in typeⅡalveolar epithelial cell and enhance the a-ENaC expression.