Pro-resolving Effects And Mechanisms Of Maresin1on Lipopolysaccharide-induced Acute Lung Injury In Mice

Part oneMaresin1inhibits neutrophil adhesion in lipopolysaccharide-induced acute lung injuryObjective:To investigate the inflammatory effects and mechanisms of Maresin1(MaRl) on the lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.Methods:One hundred and twenty eight male BALB/c mice were randomly divided into four groups:sham group, LPS group, low-dose MaRl group (LPS+LD-MaRl), high-dose MaRl group (LPS+HD-MaRl). In sham group, mice were treated with0.9%saline intravenously60mins after they were induced by intratracheal instillation of0.9%saline. In LPS group, mice were treated with0.9%saline intravenously60mins after they were induced by intratracheal instillation of LPS at dosage of3mg/kg. In LPS+LD-MaRl group, mice were intravenously treated with MaRl at dosage of0.1ng/mouse60mins after they were induced by intratracheal instillation of LPS at dosage of3mg/kg. In LPS+HD-MaRl group, mice were intravenously treated with MaRl at dosage of1ng/mouse60mins after they were induced by intratracheal instillation of LPS at dosage of3mg/kg. After24h, mice were sacrificed. Arterial blood was drawn to assess for blood gas analysis. Pulmonary pathological changes, differential leukocytes counts in broncheoalveolar fluid (BALF) and lung wet/dry ratio were observed. The protein in BALF and pulmonary myeloperoxidase (MPO) was detected by spectrophotometer. The inflammatory cytokines TNF-α, IL-1β, IL-6, KC, IL-10, MCP-5, MIP-1α and MIP-1γ in BALF were measured by ELISA kits. Neutrophilic infiltration was detected by immunohistochemistry. Neutrophil-platelet interactions were assessed by flow cytometry. The pulmonary intercellular adhesion molecule (ICAM)-1, P-selectin and CD24were evaluated by western blotting.Results:High dose of MaRl could significantly attenuate the LPS-induced lung injury and pulmonary edema, inhibit neutrophilic infiltration, and mitigate the arterial oxygenation. Meanwhile, high dose of MaRl could down-regulate the pro-inflam matory cytokines (TNF-α, IL-1β, IL-6, KC) and chemokines (KC, MCP-5, MIP-1α, MIP-1γ), and up-regulate the anti-inflammatory cytokine IL-10. Data from flow cytometry elucidated that MaRl could inhibit neutrophil-platelet interaction. Western blotting analysis revealed that MaRl decreased the expressions of P-selectin, ICAM-1and CD24.Conclusion:High dose of MaR1attenuated the inflammatory effects on the LPS-induced ALI through mitigating the pulmonary pathological changes and arterial oxygenations, decreasing the neutrophil adhesion, and down-regulating the pro-infla mmatory cytokines. Part twoPro-apoptotic effects and mechanisms of MaRl on the human neutrophilsObjective:To investigate the effects of MaRl on the human neutrophil apoptosis and explore the potential molecular mechanisms.Methods:Blood from healthy volunteers was fractioned by discontinuous density gradient of percoll to isolate the human neutrophils. Neutrophils were preincubated with MaRl (1-100nM) or/and z-VAD-fmk (20μM) for30min, and then challenged with LPS (100-500ng/ml). After24h post-LPS administration, Neutrophil apoptosis and activated caspase-3were assessed with flow cytometry. The expressions of p-ERK1/2, p-p38, p-AKT, Bcl-2and Mcl-1were analyzed by western blotting.Results:Present data suggested that MaRl at lower concentration (1-10nM) did not impact apoptosis of isolated neutrophils. However, MaRl prolonged the neutrophil survival at higher concentration (100nM). LPS suppressed the percentage of neutrophil apoptosis in a concentration-dependent manner. The concentration of LPS, which apparently inhibited the neutrophil apoptosis, was500ng/ml. Although MaRl, up to a concentration of1nM, could prevent the LPS inhibition of neutrophil apoptosis, apparent maximum inhibition was achieved at lOnM of MaRl. Meanwhile, preincubation of neutrophils with z-VAD-fink attenuated the pro-apoptotic effects of MaRl and inhibited the activation of caspase-3in neutrophils. Western blotting analysis revealed that LPS induced phosphorylation of ERK1/2, p38, AKT at30min. And MaRl protected the LPS-induced phosphorylation. Moreover, MaRl inhibited the LPS-induced expressions of Bcl-2and Mcl-1after2hours.Conclusion:This study indicated that a mechanism by which MaRl attenuated the LPS suppression of neutrophil apoptosis is inhibiting the phosphorylation of ERK1/2, p38, AKT to activate the expressions of Bcl-2and Mcl-1, and finally evoking the activation of caspase-3to mediate the apoptosis. Part threeMaRl overcomes neutrophil apoptosis and accelerates resolution of LPS-induced ALI in miceObjective:To investigate the pro-resolving effects and mechanisms of MaRl on LPS-induced ALI in mice.Methods:The experiments were divided into two parts. First, we studied whether MaRl could accelerate the resolution of the LPS-induced ALI. Eighty male BALB/c mice were randomly divided into two groups:LPS group, LPS plus MaRl group. ALI was induced by intratracheal instillation of LPS at a dosage of3mg/kg. After24h, MaRl (1ng/mouse) or saline were injected to a tail vein. Mice were sacrificed at0,1,2,4, and7day, respectively. Pulmonary pathological changes, differential leukocytes counts in BALF and lung wet/dry ratio were observed. The protein in BALF was detected by spectrophotometer. Next, we investigated whether MaRl could accelerate the resolution of the LPS-induced ALI through promoting neutrophil apoptosis. One hundred and twenty eight male BALB/c mice were randomly divided into four groups:sham group, LPS group, LPS plus MaRl group, and LPS plus MaRl and pan-caspase inhibitor group. ALI was induced by intratracheal instillation of LPS at a dosage of3mg/kg. After24hours, MaRl (1ng/mouse) or saline were injected to a tail vein. At the same time, some mice were also given intraperitoneally with the pan-caspase inhibitor z-VAD-fink (10μg/kg), followed by two additional doses of z-VAD-fink4and8h later. The saline of same volume was given to other three groups. After24h post-MaRl, Neutrophil apoptosis and activated caspase-3were assessed with flow cytometry. Pulmonary pathological changes, differential leukocytes counts and macrophage containing apoptotic bodies in BALF, and lung wet/dry ratio were observed. Pulmonary MPO activities and protein in BALF were detected by spectrophotometer. The inflammatory factors TNF-α,IL-1β, IL-10, MCP-5, MIP-1γ in BALF were measured by ELISA kits.Results:First, our results elucidated that pulmonary disturbances, neutrophil infiltration and pulmonary edema peaked at24hours post-LPS instillation. After24hours, lung injury gradually attenuated and normalized by Day7. Administration of MaRl at the inflammatory peak significantly mitigated LPS-induced ALI and pulmonary structures recovered to normal by Day4. Next, MaRl could promote neutrophil apoptosis, activate caspase-3in neutrophils, and accelerate the macrophages phagocytosing apoptotic bodies. Meanwhile, MaRl could mitigate pulmonary pathological changes, decrease the neutrophilic infiltration, reduce the pulmonary MPO activity, inhibit productions of TNF-α, IL-1β,MCP-5, MIP-1γ, and increase the production of IL-10. In contrast, pan-caspase inhibitor z-VAD-fmk could partially over-ride the pro-apoptosis of MaRl, and significantly inhibit the protective effects of MaRl. Conclusion:MaRl accelerated the resolution of the LPS-induced ALI in mice through promoting the neutrophil apoptosis.