The Protective Effect And Underlying Mechanism Of Resolvin D1 On Sepsis-induced Acute Lung Injury

BackgroundSepsis is a life-threatening critical illness caused by the maladjustment of host response to the pathogenic microbial infection,such as bacteria,fungi,viruses.It is characterized by a large amount of cytokines and inflammatory mediators,leading to systematic immune response syndrome(SIRS),septic shock,multiple organ dysfunction syndrome(MODS)or even death.Despite significant advances in understanding and treatment of sepsis,its mortality and morbidity remains high.Considering that it is the leading cause of death among critically ill patients,sepsis is a hot topic in today’s critical care medicine.The lung is frequently involved in sepsis and always the early target in the sepsis process.Acute lung injury(ALI),a manifestation of sepsis-induced complication,is secondary to SIRS and ultimately deteriorating to acute respiratory distress syndrome(ARDS),which is closely associated with high mortality.Patients with ALI and ARDS progress through a similar pathophysiological process.ALI is an early stage of ARDS,characterized by extensive inflammation and life-threatening hypoxemia,which is the result of ventilation-perfusion mismatch.In clinical,ALI is manifested with refractory arterial hypoxemia and respiratory failure with significant morbidity and mortality.ALI is caused by acute inflammation and is precipitated by the release of inflammatory cytokines,the disruption of pulmonary vascular endothelial barrier,loss of surface active substances and reduced alveolar surface tension,leakage of protein-rich fluid in the alveoli and the formation of excessive fibrosis,which results in impairment of pulmonary gas exchange between oxygen and carbon dioxide.To date,the mechanisms responsible for ALI/ARDS have not been completely delineated and it also lacks effective preventative or therapeutic approach in clinical practice.Therefore,it is of great significance to explore the pathophysiology mechanism of sepsis,especially the pathogenesis MODS and ALI/ARDS as well as seek novel effective and effective interventions for treatment and prognosis of sepsis.The resolvin D1 is a family of lipid mediators derived from eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),known as E-series resolvin(RvE)and D-series resolvin(RvD).A large number of studies have shown that resolvin has anti-inflammatory and promote the resolution of inflammation.Resolvin D1(7S,8R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid,RvD1)is processed endogenous anti-inflammatory and pro-resolution mediator from Omega-3(ω-3)fatty acid docosahexaenoic acid(DHA)。Resolvin DI could inhibit neutrophil infiltration;regulate the balance of inflammatory factors;prevention of lung fibrosis,protection of ischemia and reperfusion after organ secondary damage.Studies have shown that goblet cells secrete mucus is the main cause of ocular hypersensitivity and early dry eye,resolvin D1 can stimulate the secretion of inflammation by prophylaxis.A.nother study showed that resolvin D1 accelerate the airway mucus metaplasia in the allergic airway.The classic cecal ligation and puncture(CLP)was used to establish the animal model of sepsis to study the effect of resolvin D1 on CLP-induced lung injury in mice.The lung histopathological changes,pulmonary edema,inflammatory factors,and combined with the resolvin D1 receptor-cAMP pathway to explore the specific mechanism.In addition,the mechanism of resolvin D1 on regulating the alveolar macrophage phagocytosis,phenotype and neutrophil apoptosis to promote lung tissue inflammation was researched.Finally,we further study the effect of resolvin D1 on neutrophils on clinical sepsis patients and healthy volunteers,which could provide a new strategy for the clinical treatment of acute lung injury of sepsis.Part I The protective effect of Resolvin D1 on CLP-induced sepsis miceObjective To investigate the effect of resolvin D1 on cecal ligation and puncture(CLP)-induced sepsis mice and the possible mechanismMethods 25-30g male C57BL/6 mice were randomly divided into the following group:sham group;CLP model group,after 1%pentobarbital sodium(lmg/kg)intraperitoneal anesthesia,cecal ligation and puncture was performed,at the same time physiological saline rehydration(0.5ml/10g)to mice,then insulation blanket was used to rewarm;CLP + RvD1 group,that is,after establishment of CLP model,resolvin D1(100ng/mice)was injected through intraperitoneal;CLP + Alcohol group,which was the solvent control group(resolvin D1 dissolved in ethanol);CLP +RvD1 + BOC-2 group,after establishment of CLP model,intraperitoneal injection of resolvin D1(100ng/mice)and resolvin D1 receptor antagonist BOC-2(600ng/kg);there was 10 mice per group.The lung tissue of mice was taken at 24h and HE staining,was performed,and the lung injury scores was get to assess the injury of.lungs;The infiltration of inflammatory cells and cAMP concentration in lung tissue was observed by Elisa;The percentage of macrophages and neutrophils in the bronchoalveolar lavage fluid were collected and the ratio of macrophages to neutrophils was calculated to investigate the changes of immune cells in mice after sepsis;Mice were fed in SPF grade for 8 days to observe the survival rate.Results1.CLP model:Cecal ligation and puncture(CLP)was performed to establish the sepsis model.The mice were sacrificed 24h after CLP to HE staining.We found that the lung tissue structure was clear,the epithelial cells were arranged neatly,the alveolar cavity was clean and symmetrical in the sham group,and the lung tissue structure was obviously damaged,congestion,edema,inflammatory cell infiltration in CLP group.2.Effects of resolvin D1 on CLP-induced sepsis mice:The lung tissue structure was clear,the epithelial cells were arranged neatly,the alveolar cavity was clean and symmetrical in the sham group;While the lung tissue structure was destroyed,congestion,edema,inflammatory cell infiltration in the CLP group and the solvent control group(CLP + Alcohol)group.However,the resolvin D1(100ng)treatment group(CLP + RvD1)showed a significant improvement in lung tissue structure and decreased inflammatory cells.Compared with the sham operation group,there was no significant change in the lung tissue of the resolvin D1(RvD1)group.Lung injury scores:Compared with the sham group,the lung injury scores in CLP group,the solvent control group(CLP + Alcohol)and the resolvin D1 treatment group(CLP + RvD1)were significantly increased(P<0.05);Compared with CLP group,lung injury scores in CLP + RvD1 and RvD1 were significantly decreased(P<0.05).Wet/dry weight ratio:Compared with the sham group,the lung wet/dry weight ratio in CLP group,the solvent control(CLP + Alcohol)group were significantly increased(P<0.05),suggesting that the lung tissue edema was serious in CLP-induced lung injury;Compared with CLP group,the lung wet/dry weight ratio in CLP + RvD1 and RvD1 were significantly decreased(P<0.05);Compared with the solvent control(CLP +Alcohol)group,the lung wet/dry weight ratio in CLP + RvD1 and RvD1 were significantly decreased(P<0.05),indicating that resolvin D1 could reduced the lung edema production.3.Resolvin D1 inhibited the Inflammatory cytokines expression in lung tissue homogenates:The TNF-a,IL-1β and MPO expression in the CLP group and CLP +Alcohol group t were significantly higher than those in the sham group(P<0.05),Suggesting that inflammatory cytokines were increased after CLP injury.Compared with CLP group,the expression of TNF-a,IL-1β and MPO in CLP + RvD1 and RvD1 in lung tissue homogenates was decreased(P<0.05).The levels of TNF-a,IL-1β and MPO in the lung tissue homogenate of CLP + RvD1 and RvD1 were decreased(P<0.05)compared with the solvent control group(CLP + Alcohol),Suggesting that resolvin D1 attenuates lung injury by inhibiting the production of inflammatory factors.4.Effects of resolvin D1 on the ratio of neutrophils and macrophages in CLP-induced sepsis mice:Bronchoalveolar lavage fluid was taken to measure the neutrophils and macrophages count by flow cytometry.Compared with sham group,the ratio of macrophages to neutrophils increased(Mcarphages/Neutrophils)in CLP group(P<0.05),Suggesting the inflammatory cells are increased after CLP injury.Compared with CLP group,the ratio of macrophages to neutrophils increased(Mcarphages/Neutrophils)(P<0.05)in the resolvin D1 treatment group(CLP + RvDl),suggesting that resolvin D1 regulates the inflammatory response by regulating the ratio of macrophages to neutrophils.5.The regulation of resolvin D1 in CLP mice was dependent on its receptor,ALX:HE staining showed that the lung tissue structure in CLP group was obviously damaged,congested,edema and inflammatory cell infiltration.The resolvin D1(100ng)treatment(CLP + RvDl)group showed a significant improvement of lung tissue structure and decreased inflammatory cells,after intraperitoneal injection of BOC-2(600ng/kg)(CLP + RvD1 + BOC-2),the lung injury status was not improved.Lung injury scores:Compared with CLP group,the lung injury score in CLP + RvD1 group was significantly decreased(P<0.05);Compared with CLP + RvD1 group,CLP + RvD1 + BOC-2 group got higher lung injury score(P<0.05).Wet/dry weight ratio:Compared with CLP group,the lung wet/dry weight ratio in CLP + RvD1 was significantly decreased(P<0.05);Compared with the CLP +’RvD1 group,the lung wet/dry weight ratio in CLP + RvDl+BOC-2group was significantly decreased(P<0.05),indicating that the effect of resolvin D1 reduced the lung edema production was abrogated by its receptor inhibitor BOC-2.6.Resolvin D1 inhibited the Inflammatory cytokines expression in lung tissue homogenates in a resolvin D1 receptor dependent manner:Compared with CLP group,the expression of TNF-,IL-1β and MPO level in CLP + RvD1 group was decreased(P<0.05),suggesting that resolvin D1 attenuated lung injury by inhibiting the production of inflammatory factors;Compared with CLP + RvD1 group,the expression of TNF-ca,IL-1βand MPO in CLP + RvD1 + BOC-2 group was increased(P<0.05),suggesting that inhibitory effect of resolvin D1 on production of inflammatory factors was abrogated by BOC-2.7.Effects of resolvin D1 on the ratio of neutrophils and macrophages in CLP-induced sepsis mice was dependent on its receptor,ALX:Bronchoalveolar lavage fluid was taken to measure the neutrophils and macrophages count by flow cytometry.We found that compared with the CLP group,the ratio of macrophages to neutrophils increased(Mcarphages/Neutrophils)(P<0.05)in the CLP + RvD1 treated group(P<0.05),suggesting that the resolvin D1 increased the macrophages and neutrophils to regulate inflammatory responses;Compared with CLP + RvD1 group,the ratio of macrophages to neutrophils in CLP + RvD1 + BOC-2(600ng/kg)group was decreased(P<0.05),suggesting that the effect of resolvin D1 on regulates macrophages and Neutrophil ratio was abrogated by BOC-2.8.Effects of resolvin D1 on mice survival rate in CLP-induced sepsis mice was dependent on its receptor,ALX:Compared with sham group,the survival rate in CLP group was significantly decreased(P<0.05);Compared with CLP group,the survival rate was up-regulated in resolvin D1 treatment(CLP + RvD1)group(P<0.05);Compared with CLP + RvD1 group,the survival rate in CLP + RvD1 + BOC-2 group was down-regulated(P<0.05),suggesting that effect of resolvin D1 on survival rate in CLP was abrogated by BOC-2.9.Effects of resolvin D1 on the cAMP concentration in CLP-induced sepsis:Compared with CLP group,the cAMP concentration in CLP + RvD1 mice was increased(P<0.05);Compared with the CLP + RvD1 group,the cAMP concentration in CLP + RvD1 + BOC-2 group was decreased(P<0.05),suggesting that the resolvin D1 up-regulated the cAMP concentration in CLP mice,but this effect was suppressed by the receptor of resolvin D1 antagonist,BOC-2.Conclusion1.In this study,we established the mouse CLP model to successfully replicate the pathological process of sepsis-induced acute lung injury.2.Resolvin D1 could inhibit the expression of some inflammatory cytokines,up-regulate the ratio of macrophages to neutrophils to reduce the inflammatory response and protect lung function,then increased the survival rate of CLP mice.3.The basic effect of resolvin D1 on CLP-induced sepsis mice was abrogated by the receptor of resolvin D1 antagonist,BOC-2.Partn Ⅱ The effect of Resolvin D1 on primary macrophages and neutrophilsObjective To research the effect of resolvin D1 on primary macrophages and neutrophils in mice and to reveal the possible mechanism of resolvin D1 on regulating immune cells to promote inflammation resolution.Methods Bone marrow-derived macrophages and neutrophils were isolated from mice,various concentrations of resolvin D1(50,100,150 nM),lipopolysaccharide(LPS,1ug/ml),BOC-2(resolvin D1 receptor inhibitor)(10μM)or LY294002(PI3K inhibitor,10μM)were used to measure neutrophils apoptosis and macrophages efferocytosis.The macrophages and CMFDA Green-labeled apoptotic neutrophils at a 4:1 ratio Co-culture for 90 min to measure the efferocytosis.The effect of resolvin D1 on M2 phenotype was also detected by flow cytometry(FCM).Results1.Resolvin D1 promoted primary mouse neutrophil apoptosis:Compared with control group,neutrophil apoptosis was enhanced in LPS(1μg/ml)and LPS+ RvD1(100nM)groups(P<0.05).Compared with LPS group,neutrophil apoptosis was enhanced in LPS + RvD1 group(P<0.05).2.Resolvin D1 dose-dependently promoted macrophage efferocytosis:Compared with the control group,resolvin D1 dose-dependently increased macrophage efferocytosis(P<0.05),and the best concentration of resolvin Dlwas 100nM.3.Resolvin D1 improved macrophage efferocytosis in a receptor and PI3K-dependent manner:Compared with the control group,the efferocytosis rate of macrophages decreased in LPS(1μp/ml)group(P<0.05);Compared with LPS group,the efferocytosis rate of macrophages in LPS + RvD1 group was increased(P<0.05);Compared with LPS + RvD1 group,macrophage phagocytosis was decreased in LPS + RvD1 + BOC-2(10 μM)group and LPS + RvD1 + LY(LY294002,10 μM)group(P<0.05).4.Resolvin D1 promoted efferocytosis of BALF-stimulated macrophages:Compared with the control group,the efferocytosis of macrophages in BALF stimulation group was decreased(P<0.05),and the efferocytosis of resolvin D1 group was higher(P<0.05);Compared with the BALF group,macrophage efferocytosis was increased in RvDl + BALF group(P<0.05).5.Resolvin D1 regulated macrophage efferocytosis in a receptor and PI3K-dependent manner:Compared with the control group,the phosphorylation of phospho-PI3K P85(P-P85)protein expression in LPS group was decreased(P<0.05),and the expression of P-P85 in resolvin D1 group was increased(P<0.05);Compared with LPS group,the expression of P-P85 protein in RvD1 + LPS group was increased(P<0.05);Compared with RvD1 + LPS group,the expression of P-P85 was decreased after BOC-2 intervention(P<0.05).There was no significant difference in the expression of phosphatidylinositol kinase PI3K P85(P85)protein in each group(P>0.05).6.Resolvin D1 up-regulated the expression of M2 macrophages phenotype:Compared with the control group,the expression of M2 phenotype in LPS group was decreased,and the level of M2 phenotype in resolvin D1 group was increased;Compared with LPS group,M2 phenotype in LPS + RvD1 group was increased.Conclusion1.Resolvin D1 promoted inflammation resolution through improving neutrophil apoptosis2.Resolvin D1 promoted inflammation resolution through enhancing macrophage efferocytosis and increasing M2 phenotype expression3.Resolvin D1 promoted macrophage efferocytosis was associated with resolvin D1 receptors and P-P85Part Ⅲ The effect of Resolvin D1 on Healthy and Sepsis Patient NeutrophilsObjective To study the effect of resolvin D1 on neutrophils in sepsis patients and to explore its possible mechanism.Methods Twenty healthy volunteers and 40 sepsis patients were involved in this study.Neutrophils were isolated from Peripheral blood.Pre-stimulated neutrophils with resolvin D1 for 1h to measure chemotaxis,apoptosis,pagocytosis,extracellular trapping(NETs),reactive oxygen species(ROS),cell surface adhesion molecule expression and COX-2,NF-κB protein expression.To compare the effect of resolvin D1 on neutrophils in healthy volunteers and sepsis patients and reveal its possible mechanism.Results1.Resolvin D1 inhibited neutrophil chemotaxis:Healthy volunteers:Compared with control group,IL-8 and fMLP enhanced neutrophil chemotaxis index and migration rate(Velocity);Compared with IL-8 group,the chemokine index and the migration rate(Velocity)were inhibited in RvD1-IL-8 group(P<0.05);Compared with the fMLP group,the chemotaxis index and the migration rate(Velocity)were inhibited in RvD1-fMLP group(P<0.05).Sepsis patients:Because of neutrophil dysfunction,the cells did not occur significantly migration.2.Resolvin D1 promoted neutrophil apoptosis:Compared with control group,the apoptotic cells(Dying)increased(P<0.05)after resolvin D1(100nM)stimulated for 4h and 24h,suggesting that resolvin D1 promoted neutrophil apoptosis in healthy volunteers and sepsis patients.At 24h,apoptotic cells in sepsis patients were more than volunteers,suggesting that neutrophil apoptotic was delayed in sepsis;Compared with the resolvin D1 group,the apoptosis of neutrophils were decreased after 4h intervention with BOC-2(1uM)(P<0.05),but the apoptotic neutrophils were not significantly changed after stimulated with PI3K inhibitor LY294002(1uM)for 4h(P>0.05),suggesting that resolvin D1 promoted neutrophil apoptosis dependent on its receptor,ALX.3.Resolvin D1 improved neutrophils NETs production:The neutrophil NETs production in sepsis patients were less than those in the volunteers group,but there was no significant difference(P>0.05).After PMA(25 nm)intervention,the formation of NETs in sepsis PMA group was significantly higher than that in healthy PMA group(P<0.05);Compared with control group,the formation of NETs was increased after PMA intervention(P<0.05),and the production of NETs was increased in RvD1-PMA group(P<0.05);Compared with the resolvin D1 group,the formation of NETs was increased in the RvD1-PMA group(P<0.05),suggesting that resolvin D1 improved NETs formation.4.Resolvin D1 promoted neutrophil phagocytosis:The ability of neutrophils to phagocytose E.coli and S.aureus increased in a time dependent manner,and peaked at 60 min.At 15 min and 30 min,the ability of neutrophils to phagocytose E.coli in sepsis patients was lower than that in volunteers(P<0.05);Compared with the volunteers group,phagocytosis was increased at 30 min and 45 min in RvD1(100 nM)group;Compared with the sepsis group,phagocytosed E.coli was increased at 30 min and 45 min(P<0.05)and phagocytosed S.aureus was increased at 60 min in RvDl(100 nM)group(P<0.05),Suggesting that resolvin D1 promoted neutrophil to phagocytose E.coli and S.aureus.5.Resolvin D1 inhibits the production of reactive oxygen species(ROS):Compared with the control group,the ROS production in the PMA group and fMLP group was decreased(P<0.05);Compared with RvD1 group,the ROS production in the RvD1+PMA group or RvD1 + fMLP group was significantly higher(P<0.05);Compared with the PMA group,the expression of ROS in RvD1+ PMA group was lower(P<0.05);ROS production under PMA stimulated in sepsis group was higher than volunteers(P<0.05),suggesting that resolvin D1 inhibits healthy volunteers and sepsis patients with neutrophil ROS production.6.Effects of resolvin D1 on neutrophil surface adhesion molecules in healthy volunteers and sepsis patients:There was no significant difference in the expression of surface adhesion molecules in the volunteers groups(P>0.05);In sepsis patients,compared with the control group,the expression of CD62L was increased(P<0.05),and the expression of CD14L was significantly lower in the RvD1 group(P<0.05),suggesting that resolvin D1 inhibited the expression of CD 14 and promotes the expression of CD62L on the surface of neutrophils in sepsis patients;Compared with the volunteers group,the expression of CD14 and CD63 in sepsis patients was increased(P<0.05),and the expression of CD62L was decreased(P<0.05).7.The expression of COX-2 and NF-κB in neutrophils was regulated by resolvin D1:Compared with control group,the expression of COX-2 and NF-κB/P-P65 protein in neutrophils was decreased(P<0.05)in RvD1 group in healthy volunteers and sepsis patients;Compared with healthy volunteers,the expression of COX-2,NF-κB/P-P65 and NF-κB/P65 protein in sepsis patients was increased(P<0.05).Conclusion1.Resolvin D1 promoted inflammation resolution through inhibiting the neutrophil chemotaxis,promoting apoptosis,enhancing phagocytosis,inhibiting reactive oxygen production and regulating cell surface markers of molecules expression.2.Resolvin D1 inhibited the expression of COX-2 and NF-κB/P-P65 in neutrophils suggesting resolvin D1 promoted inflammation resolution through NF-κB signaling pathway.3.The role of resolvin D1 in patients with sepsis was stronger than healthy volunteers,suggesting that resolvin D1 was associated with inflammation.