Protective Effects Of BML-111, A Lipoxin Receptor Agonist, On Ventilator-induced Lung Injury In Rats

Part I Effects of BML-111on the inflammatory responses following ventilator-induced lung injury in ratsObjective To investigate the effects of different dose lipoxin receptor agonist BML-111on the inflammatory responses following ventilator-induced lung injury in rats.Method Forty eight male Sprague-Dawley(SD) rats of6-8weeks old, weighing200-250g, were randomly divided into six groups(n=8):sham group (sham), underwent tracheotomy but breathed spontaneously; low tidal volume group (LVT), tidal volume6ml/kg; high tidal volume group (HVT), tidal volume20ml/kg; O.lmg/kg BML-111treatment group(BML(0.1)), tidal volume20ml/kg, treated with BML-111(0.1mg/kg) intraperitoneally at the beginning of ventilation; lmg/kg BML-111treatment group (BML(1)), tidal volume20ml/kg, treated with BML-111(1mg/kg) intraperitoneally at the beginning of ventilation; BOC-2plus BML-111treatment group (BOC+BML(1)), tidal volume20ml/kg, treated with BML-111(1mg/kg, intraperitoneally) at the beginning of ventilation, BOC-2(50μg/kg, intraperitoneally) was administered30min before ventilation. Rats with mechanical ventilation were ventilated either at6ml/kg with80breaths/min or at20ml/kg with80breaths/min for4h. PaO2levels were determined immediately by blood gas analyzer at the end of experiment, and rats were sacrificed by exsanguination. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Histological changes were scored under light microscope according to the lung injury levels. Lung wet/dry weight ratio (W/D) and myeloperoxidase (MPO) activity were determined. Meanwhile, differential cell counts were performed on Wright-Geimsa-stained cytocentrifuged preparations of rats’ BALF. BALF protein concentration was measured with a BCA kit. BALF and lung tissue levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1β,IL-6, IL-10and monocyte chemoattratctant protein (MCP)-1were determined by enzyme-linked immunosorbent assay (ELISA).Results Compared with the sham Group, the alveolar structure obviously changed in the HVT Group with a significantly higher lung injury score (P<0.05); the PaO2levels significantly reduced (P<0.05); pulmonary MPO activity and W/D markedly elevated (P<0.05); the total cell, neutrophils and monocytes/macrophages counts were increased (P<0.05) in the BALF; the concentration of TNF-a, IL-1β, IL-6, IL-10and MCP-1in the BALF and lung tissue were highly up-regulated (P<0.05). However, compared with the HVT Group, PaO2were significantly elevated (P<0.05) in the BML (1) Group; whereas there were no statistially significant difference of IL-10concentration in both BALF and lung tissue; lung injury score, pulmonary MPO activity, W/D, the total cell, neutrophils and monocyte/macrophage counts, the concentration of TNF-a, IL-1β, IL-6and MCP-1in the BALF and lung tissues were declined significantly (P<0.05).Conclusion BML-111(lmg/kg) can improve the pathological changes of ventilator-induced lung injury with reduction of neutrophil infiltration and pro-inflammatory cytokines expression in BALF and lung tissues. BML-111played a protective role on ventilator-induced lung injury in rats. Part Ⅱ The effects of BML-111on NF-κB and MAPK/AP-1signaling pathways following ventilator-induced lung injury in ratsObjective To investigate whether BML-111exerts protective effects in ventilator-induced lung injury in rats by inhibiting nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK)/activator protein-1(AP-1) signaling pathways. Method Thirty two male SD rats,6-8weeks old, bodyweight200-250g, were used in this study. Animals were randomly divided into four groups (n=8):low tidal volume group (LVT), tidal volume6ml/kg; high tidal volume group (HVT), tidal volume20ml/kg; lmg/kg BML-111treatment group (BML(1)), tidal volume20ml/kg, treated with BML-111(1mg/kg) intraperitoneally at the beginning of ventilation; BOC-2plus BML-111treatment group (BOC+BML(1)), tidal volume20ml/kg, treated with BML-111(1mg/kg, intraperitoneally) at the beginning of ventilation, BOC-2(50μg/kg, intraperitoneally) was administered30min before ventilation. Rats with mechanical ventilation were ventilated either at6ml/kg with80breaths/min or at20ml/kg with80breaths/min for4h. NF-κB in the cytoplasmic and nucleus protein fraction of lung tissue was evaluated by Western blotting. And inhibitor of κB-a (IκB-a) in the cytoplasmic protein fraction of lung tissue was detected in the same way. Total and phosphorylation of ERK, p38MAPK and JNK in the lung tissues were also evaluated by Western blotting. DNA-binding activity of NF-κB and activator protein-1(AP-1) were measured by electrophoretic mobility shift assay (EMSA).Results The expression of IκB-α significantly decreased in response to high tidal volume ventilation (P<0.05). The expression of IκB-α in the cytoplasmic protein was significantly up-regulated by the application of lmg/kg BML-111(P<0.05). Whereas the IκB-a expression was down-regulated while pretreatment with BOC-2(P<0.05). High tidal volume ventilation obviously increased the cytoplasm-to-nucleus translocation of NF-κB (P<0.05). On the contrary, administration with lmg/kg BML-111significantly inhibited the nuclear translocation of NF-κB (P<0.05). And this effect was blocked by BOC-2pretreatment (P<0.05). High tidal volume ventilation resulted in increased ERK, p38MAPK and JNK phosphorylation compared with the low tidal volume ventilation (P<0.05). High tidal volume ventilation induced ERK, p38and JNK phosphorylation were significantly attenuated in the BML(1) group (P<0.05). The BOC+BML(1) group animals had similar ERK, p38and JNK phosphorylation levels as the HVT group animals (P< 0.05). The deoxyribonuclease-binding activity of NF-κB and AP-1in pulmonary significantly increased in the HVT animals. This change was reduced in the BML(1) group. Whereas, the deoxyribonuclease-binding activity of NF-κB and AP-1were markedly up-regulated in the BOC+BML(1) group.Conclusion BML-111was found to increase the expression of IκB-a in the cytoplasm and decrease the translocation of NF-κB from cytoplasm to nucleus. BML-111markedly inhibited VILI-induced phosphorylation of ERK, p38MAPK and JNK, blocked VILI-induced DNA-binding activity of NF-κB and AP-1. The results suggest that BML-111exerts protective effects at least partly through blocking NF-κB and MAPK/AP-1signaling transduction pathway in VILI in rats. Part Ⅲ Lipoxin receptor agonist BML-111accelerated the resolution of ventilator-induced lung injury via promoting the neutrophilic apoptosis in ratsObjective To investigate the pro-resolving effects and mechanism of BML-111on ventilator-induced lung injury in rats.Methods The experiments were divided into two parts. In the first set of experiments, we investigated whether BML-111could accelerate the resolution of VILI. One hundred and four SPF grade SD rats,6-8weeks, weighing200-250g, were divided into two groups:ventilator-induced lung injury group (VILI), tidal volume20ml/kg,1h; VILI plus BML-111treatment group (BML-111), tidal volume20ml/kg, lh, BML-111(1mg/kg) intraperitoneally12h after ventilation. In VILI group, animals were sacrificed at Oh,6h,12h,24h,48h,3d,4d and7d after mechanical ventilation, respectively. In BML-111group, animals were sacrificed at24h,48h,3d,4d and7d after mechanical ventilation. Histological changes in lung tissue were observed under an optical microscope. Lung wet to dry weight ratios (W/D) were determined. After Wright-Geimsa stain, the differential cell counte was determined. The total concentration of BALF protein was measured by using the BCA protein assay kit. The expression levels of tumor necrosis factor (TNF)-a, interleukin (IL)-1β, IL-6, and IL-10in BALF were determined by ELISA. The second part of experiment were designed to study whether BML-111accelerate the resolution of VILI through promoting the neutrophil apoptosis. Forty SPF grade SD rats,6-8weeks, weighing200-250g, were divided into five groups:sham group (sham), rats were orally intubated but breathed spontaneously; ventilator-induced lung injury group (VILI), tidal volume20ml/kg,1h; VILI plus BML-111treatment group (BML-111), tidal volume20ml/kg,1h, BML-111(1mg/kg) intraperitoneally12h after ventilation; VILI+pan-caspase inhibitor z-VAD-fmk treatment group (z-VAD), tidal volume20ml/kg,1h, z-VAD-fink (10μg/kg), intraperitoneally three times at4h intervals; VILI+BML-111+pan-caspase inhibitor z-VAD-fmk treatment group (z-VAD-BML), tidal volume20ml/kg,1h, BML-111(1mg/kg) intraperitoneally12h after ventilation, z-VAD-fmk (10μg/kg), intraperitoneally three times at4h intervals. Twenty four hours after mechanical ventilation, PaO2levels were detected and animals were sacrificed. Neutrophil apoptosis were assessed with flow cytometry. Histological changes were scored under light microscope according to the lung injury levels. Lung W/D was determined. The differential cells were counted after Wright-Geimsa stain. Total protein concentration in BALF was assayed with a BCA kit. Levels of TNF-a, IL-1β, IL-6, and IL-10in BALF were quantified by ELISA.Results Mechanical ventilation caused severe lung injury, with maximal pulmonary inflammatory response seen at12h, and resolution of inflammation completed at3d after VILI. Treatment with BML-111accelerated the resolution of VILI. Resolution of inflammation completed at2d after VILI. In the second part, BML-111promoted the neutrophil apoptosis, mitigated pulmonary pathological changes, reduced the neutrophilic infiltration, decreased the levels of the TNF-a, IL-1β,IL-6, increased the production of the pro-resolving cytokine IL-10. In contrast, pan-caspase inhibitor z-VAD-fmk significantly inhibited the protective effects of BML-111.Conclusion BML-111accelerated the resolution of the VILI in rats through promoting the neutrophil apoptosis.