Protective Effects And Mechanisms Of Aspirin-triggered Lipoxin A₄on Lipopolysaccharide-induced Acute Lung Injury In Mice

Part oneThe model of LPS-induced acute lung injury in miceObjective: To explore a reliable, minimally invasive and efficient method for endotracheal intubation, and instill lipopolysaccharide (LPS) to induce acute lung injury (ALI) in mice.Methods: Forty male BALB/C mice were randomly distributed into two groups (n=20/group):lipopolysaccharide group (LPS) and normal saline group (NS). After successfully endotracheal intubation, mice were instilled of LPS (3mg/kg) or NS (1.5ml/kg). Record the once success rate, final success rate of the endotracheal intubation and survival rate. At24hours after instillation, artery blood gas was tested. Wet/dry weight ratio (W/D ratio) was determined after the lung became dry. The histopathology of lung tissue was observed under a light microscope. Total cells in bronchoalveolar lavage fluid (BALF) were counted under a light microscope. The differential cells were counted after Wright-Geimsa stain. Total protein concentration in BALF was assayed with a BCA kit. Levels of tumor necrosis factor-alpha (TNF-a) and interleukin-6(IL-6) in BALF were quantified by ELISA.Results:The once success rate and final success rate of endotracheal intubation in mice were92.5%and100%, respectively. Survival rate of mice in the whole study was100%. PaO2of artery blood and Oxygenation index both decreased in LPS group in comparison to NS group (P<001). W/D ratio of lung tissue in LPS group was significantly elevated compared with NS group (P<0.001). Under a light microscope, it was observed that serious alveolar atelectasis, thickening of alveolar wells, significant neutrophil infiltration in the lung intersririal and alveoli, servere pulmonary interstitial edema, hemorrhage and alveolar congestion appeared in the lung tissues of LPS group mice. No pathological changes were presented in the lung tissue of NS group. Compared with NS group, the total cells and polymorphonulear leukocytes markedly increased (P<0.001), whereas there was no statistially significant difference of mononuclear leukocytes between the two groups. Total protein concentration, TNF-a level and IL-6level in BALF of LPS group were higher than these in BALF of NS group (P<0.05).Conclusion:Improved method for endotracheal intubation has high success rate and minimal injury, as well as instillation of LPS (3mg/kg) can induce ALI in mice successfully. Part twoEffects of ATL on the inflammation responses in LPS-induced ALI in miceObjective: The study aimed to investigate whether ATL alleviates excessive inflammation responses in LPS-induced ALI in mice.Methods: Eighty male BALB/C mice were randomly divided into four groups (n=20/group):control group, ALI group, ATL1μg-LPS group and ATL5μg-LPS group. Mice were instilled intratracheally with LPS (3mg/kg body weight) to induce ALI and endotoxin-free saline (1.5ml/kg body weight) as a control. ATL (1μg or5μg) or vehicle was injected via tail-vein injection30min prior to LPS instillation. After24hours of instillation, artery blood gas was tested for oxygenation index. W/D ratio was determined after all lung lobes became dry. Under a microscope, the histology changes of lung tissue were observed and then lung injure scores were quantified. Total cells, polymorphonuclear and mononuclear leukocytes in BALF were counted under a light microscope. Total protein concentration in BALF was assayed by using a BCA kit. Pulmonary myeloperoxidase (MPO) activity was measured by a MPO kit. Levels of TNF-a, IL-6, monocyte chemoattractant protein-1(MCP-1) and interleukin-10(IL-10) in BALF were analysed by ELISA.Results:Compared with control group, mice in ALI group had lower oxygenation index (P<0.01), higher W/D ratio and lung injury score (P<0.01). Total cells counts, polymorphonuclear and mononuclear leukocytes all markedly increased in ALI group (P<0.01). Pulmonary MPO activity was enhanced in ALI group (P<0.01). Total protein concertration and levels of pro-inflammatory cytokines (TNF-a, IL-6and MCP-1) in BALF of ALI group were significantly upregulated (P<0.01), whereas anti-inflammatory cytokine IL-10was downregulated (P<0.01). In comparision with ALI group, mice with ATL adminiatration had higher oxygenation index and lower W/D ratio (P<0.05). ATL markedly improved the lung histopathology with a lower lung injury score in contrast to ALI group (P<0.01). ATL led to a dose-dependent decrement in LPS-induce total cells counts, polymorphonuclear leukocytes and total protein concentration (P<0.05), but had no effect on mononuclear leukocytes. Only5μg ATL significantly decreased MPO activity of lung tissue compared with ALI group (P><0.05). ATL significantly reduced LPS-induced TNF-a, IL-6, and MCP-1levels in BALF (P<0.05), whereas1μg ATL dramatically promoted IL-10expression (P<0.01).Conclusion: This study suggests that exogenous ATL attenuates LPS-induced ALI and inflammation reponses in mice by improving lung oxygenation and histology, enhancing IL-10production, inhibiting LPS-induced MPO activity, leukocytic infiltration, TNF-a, IL-6and MCP-1. Part threeEffects of ATL on MAPK/AP-1and NF-κB signalling pathways following acute lung injury in miceObjective:In order to investigate whether ATL exerts protective effects in LPS-induce ALI in mice by inhibiting LPS-activated mitogen-activated protein kinases (MAPKs)/activator protein-1(AP-1) and nuclear factor-kappa B (NF-κB) signalling pathways.Methods: Thirty male BALB/C mice were randomly divided into three groups (n=10/group):control group, ALI group and ATL5μg-LPS group. Mice were intratracheally instilled with LPS (3mg/kg body weight) to induce ALI and endotoxin-free saline (1.5ml/kg body weight) as a control.5μg ATL or vehicle was injected via tail-vein injection30min before LPS instillation. After24hours of instillation, total and phosphorylations of p38, ERK1/2and JNK in the lung tissue were determined by Western blotting. NF-κB p65and inhibitor of κB-α (IκB-α) in the cytoplasmic protein fraction of lung tissue were also measured by Western blotting. DNA-binding activity of NF-κB p65and activator protein-1(AP-1) were assayed by electrophoretic mobility shift assay (EMSA).Results:Compared with control group, phosphorylations of p38MAPK, ERK1/2and JNK in the lung tissue were significanly enhanced in ALI group (P<0.01), whereas5μg ATL markedly inhibited LPS-induced phosphorylations of p38, ERK1/2and JNK (P<0.05). NF-κB p65and IκB-α in the cytoplasmic protein fraction of lung tissue were significant less than those in control group (P<0.05). Pretreatment with5μg ATL kept higher NF-κB p65and IκB-α expression in the cytoplasmic protein fraction of lung tissue in contrast to ALI group(P<0.05). Moreover, EMSA showed that DNA-binding activity of AP-1and NF-κB p65were increased in ALI group compared with control, whereas5μg ATL administration dramatically decreased LPS-induced DNA-binding activity of AP-1and NF-κB p65.Conclusion:ATL markedly inhibited LPS-induced phosphorylations of p38MAPK, ERK1/2and JNK, enhanced IκB-a expression in the cytoplasm, decreased the translocation of the NF-κB p65from the cytoplasm into the nucleus, blocked LPS-induced DNA-binding activity of AP-1and NF-κB p65. The current study illustrates that ATL exerts protective effects at least partly through blocking NF-κB and MAPKs/AP-1signaling transduction pathway in LPS-induced ALI in mice.