The Effect And Mechanism Of Lipoxin A4 On Neutrophil Function Of Acute Respiratory Distress Syndrome(ARDS)

Background:Acute respiratory distress syndrome(Acute Respiratory Distress Syndrome,ARDS)is a common clinical emergency.In recent years,although the research on the pathogenesis of ARDS has made great progress,there is still a lack of specific treatment.Therefore,it is urgent to study the pathogenesis and treatment of ARDSNeutrophils are the main effector cells of the innate immune system,which play an important role in the occurrence,development and resolution of inflammation.In the process of ARDS disease,activated neutrophils play an important role in the clearance of ARDS inflammation by gathering from the peripheral circulation to the lung tissue,and eliminating potential harmful stimuli in ARDS patients.However,neutrophils are also highly histotoxic cells.Excessive accumulation of neutrophils in pulmonary microcirculation,pulmonary interstitium and alveolar spaces in ARDS patients will cause neutrophil-mediated tissue damage.Therefore,how to effectively promote the antimicrobial activity of neutrophils and accelerate the resolution of dead neutrophils has always been our concern.Neutrophils need to maintain their homeostasis through appropriate ways of death to avoid autoimmune diseases or other inflammatory diseases.Neutrophils die in many ways,among which the most unique biological behavior of neutrophils is the formation of "neutrophil extracellular traps(NETs)" and subsequent death NETosis.NETs is mainly composed of loose chromatin and granule(source)proteins on it.The formation of NETs requires the participation of neutrophil elastase(NE)and myeloperoxidase(MPO).Studies have confirmed that the trapping effect of NETs can effectively protect the body from pathogen infection,but neutrophils trap and kill pathogenic microorganisms accompanied by their own death(NETosis),which is a different kind of tissue damage.Therefore,effective enhancement of neutrophil NETs function and timely removal of NETosis-related tissue damage is the key to promote the regression of ARDS inflammation.Lipoxin A4(LXA4)is an important endogenous anti-inflammatory and pro-resolution mediator produced in the process of inflammatory resolution,which plays an important role in the process of inflammation resolution.LXA4 not only has strong functions of anti-inflammatory and promoting inflammatory resolution,but also can regulate the body’s immune function and promote the repair of damaged cells and tissues.However,the level of LXA4 in ARDS patients,the protective effect of exogenous LXA4 on ARDS patients and its specific mechanism are still unclear.Therefore,in this study,the peripheral blood of ARDS patients was collected and the content of LXA4 was detected by UPLC-MS technique,the mRNA expression of LXA4 receptor(ALX receptor)and key synthetic enzymes(5-LOX,15-LOX-1)in peripheral blood leukocytes were detected by RT-PCR.Peripheral blood neutrophils were isolated from patients,and the effect of LXA4 on the function of peripheral blood neutrophils in ARDS patients was detected.Finally,ARDS rat models were used to further explore the specific mechanism of LXA4 promoting the resolution of inflammation and affecting the neutrophil extracellular traps(NETs)function in ARDS rats by affecting the function of neutrophils.Part Ⅰ:The expression of Lipoxin A4 in patients with ARDSObjective:The purpose of this part of the study was to detect the expression LXA4 in peripheral blood of ARDS patients,and to observe the mRNA expression levels of LXA4 receptor(ALX receptor),synthase 5-LOX and 15-LOX-1 in peripheral blood leukocytes of ARDS patients.Materials and methods:1.Peripheral blood samples were collected from 10 patients with ARDS and 10 healthy volunteers.We extracted plasma from peripheral blood sample and detected the expression of LXA4 in peripheral blood of ARDS patients and healthy volunteers by ultra high performance liquid chromatography-mass spectrometry(UPLC-MS)after solid phase extraction.2.RT-PCR technique was used to compare the mRNA expression levels of LXA4 receptor(ALX receptor)and key synthetic enzymes(5-LOX,15-LOX-1)in peripheral blood leukocytes of patients with ARDS and healthy volunteers.Results:1.The results of UPLC-MS test showed that the concentration of LXA4 in ARDS patients was significantly lower than that in healthy volunteers(P<0.01).2.The results of RT-PCR detection showed that the level of ALX receptor on peripheral leukocytes in ARDS patients was significantly lower than that in healthy volunteers(P<0.01).3.The results of RT-PCR test showed that compared with the healthy control group,the levels of 5-LOX and 15-LOX-1 in the ARDS patients were decreased(P<0.05),and the decrease of 15-LOX-1 was more obvious(P<0.01),and the difference was statistically significant.Conclusions:The content of LXA4 in ARDS patients is very low,and the expression of its related synthase 5-LOX and 15-LOX-1 and its receptor ALX in ARDS patients is also significantly lower than that in healthy volunteers,suggesting that there may be inflammation resolution disorder in ARDS patients.Part Ⅱ:The effect of Lipoxin A4 on the function of peripheral blood neutrophils in ARDS patientsObjective:This part of the study is based on the functional studies of neutrophil sterilization,phagocytosis,neutrophil extracellular traps(NETs),apoptosis and so on,to explore the mechanism of the effect of LXA4 on the function of peripheral blood neutrophils in ARDS patients,and to provide a new possible method for the treatment of ARDS.Materials and methods:1.Peripheral blood neutrophils were isolated from ARDS patients and healthy volunteers,and the purity and vitality of neutrophils were identified by Wright staining.The best concentration of LXA4 on neutrophils was explored by CCK-8 experiment.2.Peripheral blood neutrophils from ARDS patients were separated and divided into four groups:(1)Control group;(2)LXA4 group;(3)LXA4 receptor antagonist(BOC-2)group;(4)LXA4+BOC2 group.We detected the effects of LXA4 on neutrophil reactive oxygen species(ROS)production by Luminometer instrument,and detected the effect of LXA4 on neutrophil extracellular traps(NETs)function by Fluorescence Microplate Reader.Flow Cytometry was used to detect the effects of LXA4 on neutrophil phagocytosis,apoptosis and NETosis death.Results:1.Neutrophils from peripheral blood of ARDS patients were extracted by percol density gradient centrifugation.The purity of neutrophils was(97.5±2.3)%,and the cell viability was≥96%.2.Different concentrations of LXA4 stimulated neutrophils for 4 hours.CCK-8 results showed that 10 nM,50 nM and 100 nM LXA4 could be used as neutrophils stimulation doses.Combined with the effects of different concentrations of LXA4 on peripheral blood neutrophils respiratory burst and reactive oxygen species(ROS)production,we finally determined that 100 nm LXA4 had the best effect in stimulating peripheral blood neutrophils.3.The results of Fluorescence Microplate Reader showed that the content of NETs produced by the patients with ARDS was higher than that of healthy volunteers(P<0.05),and ROS production of neutrophil was significantly higher in ARDS patients than in healthy volunteers(P<0.01).LXA4 can promote the production of NETs in peripheral blood neutrophils of ARDS patients in vitro by binding to ALX receptor.4.Luminometer test results showed that the ROS content of ARDS patients without stimulation was higher than that of healthy volunteers(P<0.01).After PMA stimulation,the ROS production of peripheral blood neutrophils of ARDS patients was significantly higher than that of healthy volunteers(P<0.01).However,LXA4 can inhibit the production of ROS in peripheral blood neutrophils of ARDS patients by binding to ALX receptor.5.The results of Flow Cytometry showed that the phagocytic ability of S.aureus by neutrophils in peripheral blood of ARDS patients was higher than that of healthy volunteers at 30 min,45 min and 60 min(P<0.05),and the phagocytic ability of E.coli by neutrophils in peripheral blood of ARDS patients was higher than that of healthy control group at 45 min and 60 min(P<0.05).LXA4 could not only enhance the phagocytic ability of S.aureus by neutrophils in peripheral blood of ARDS patients(P<0.05),but also enhance the phagocytic ability of E.coli by neutrophils in peripheral blood of ARDS patients(P<0.05).6.Flow Cytometry results showed that LXA4 could promote the apoptosis of peripheral blood neutrophils of ARDS patients cultured for 4 hours and 24 hours in vitro(P<0.05),and significantly reduce the death of NETosis of peripheral blood neutrophils of ARDS patients cultured for 24 hours in vitro(P<0.01).The effect of LXA4 on neutrophil apoptosis and NETosis death is mediated by ALX receptor.Conclusion:1.LXA4 enhances the bactericidal function of peripheral blood neutrophils in ARDS patients by enhancing the ability of neutrophil extracellular traps(NETs)and phagocytosis.2.LXA4 can reduce the production of ROS in peripheral blood neutrophils of ARDS patients and alleviate the damage of neutrophils to surrounding tissues.3.LXA4 can promote neutrophil apoptosis in peripheral blood of ARDS patients,reduce the death of NETosis and accelerate the inflammatory resolution of ARDS.Part Ⅲ:The protective effect and mechanism of lipoxin A4 on ARDS rats by affecting the function of peripheral blood neutrophilsObjective:In this study,we used animal and cell experiments to explore the lung protective effect of LXA4 on ARDS rat models by affecting the function of neutrophils.Materials and methods:1.LPS(14 mg/kg)was administered intravenously for 6 hours to establish LPS induced ARDS rat model.The rats were divided into 4 groups and 6 rats in each group:(1)Control group:equal volume of normal saline was injected into caudal vein,(2)LPS group:14 mg.kg LPS was injected into caudal vein after 6 hours;(3)LXA4 group:the same volume of normal saline was injected into the caudal vein of the treatment group 6 hours later,LXA4 200 ng/kg was injected through the caudal vein,(4)LPS+LXA4 group:after the tail vein injection of 14 mg/kg LPS for 6 hours,and LXA4 200 ng/kg was injected through the tail vein.24 hours after the establishment of the model,the blood was taken from the heart,the rats were killed,the lung tissue was taken,the pathological changes were observed by HE staining,the lung injury score was scored,and the levels of TNF-α and IL-1β in the blood of mice in each group were detected by ELISA.2.Intraperitoneal injection of cyclophosphamide(75 mg/kg)4 days and 1 day before LPS induced ARDS rats was used to establish the ARDS rat models of neutropenia.They were divided into three groups with 6 rats in each group:(1)Control group,(2)LPS group;(4)LPS+LXA4 group(the same dosage as before).24 hours after successful modeling,the blood was taken from the heart,the rats were killed,the lung tissue was taken,the pathological changes were observed by HE staining,the lung injury score was made,and the levels of TNF-α and IL-1β in the lung tissue homogenate of each group were detected by ELISA.3.Flow Cytometry was used to separate neutrophils from peripheral blood of rats,and immunofluorescence staining was used to identify their purity and activity.4.Peripheral blood neutrophils of ARDS rats were separated by Sorting Flow Cytometry and divided into four groups:(1)Control group;(2)LXA4 group;(3)LXA4 receptor antagonist(BOC-2)group;(4)LXA4+BOC-2 group.The effect of LXA4 on the function of neutrophil extracellular traps(NETs)was detected by Fluorescence Microplate Reader,and the effect of LXA4 on neutrophil apoptosis and NETosis in ARDS rats was detected by Flow Cytometry.5.The neutrophils were separated by Sorting Flow Cytometry and divided into five groups:(1)Control group,(2)LXA4 group,(3)LXA4+NE inhibitor(NEi)group,(4)LXA4+MPO inhibitor(MPO inhibitor I)group,(5)LXA4+NEi+MPO inhibitor I group.Western blot was used to detect the effect of LXA4 on the content of NE and MPO in neutrophils,and Fluorescence Microplate Reader was used to detect the effect of LXA4 on the function of neutrophil extracellular traps(NETs).Results:1.Exogenous administration of LXA4 could significantly reduce the degree of lung injury in ARDS rats(P<0.07),and significantly reduce the levels of pro-inflammatory cytokines TNF-α and IL-1β in lung tissue homogenate(P<0.01),but LXA4 had no lung protective effect on ARDS rats with neutropenia(P>0.05),and could not reduce the levels of pro-inflammatory cytokines TNF-α and IL-1β in lung tissue homogenate(P>0.05).2.Sorting Flow cytometry was used to extract neutrophils from rat peripheral blood.Immunofluorescence staining showed that the purity of neutrophils was 100%and the cell viability was more than 95%.3.The results of Fluorescence Microplate Reader showed that LXA4 could promote the production of NETs in vitro by binding to ALX receptor(P<0.05).4.Western blot results showed that the amount of NE and MPO released by LPS group was higher than that of control group(P<0.05),and LXA4 could further promote the release of NE and MPO from neutrophils(P<0.01).5.The results of Fluorescence Microplate Reader showed that NE inhibitor(NEi)and MPO inhibitor I could block the promoting effect of LXA4 on NETs(P<0.01),and the synergistic effect of combination of them was greater than that of single use(P<0.01).6.The results of Flow Cytometry showed that LXA4 promoted the apoptosis of neutrophils in ARDS rats cultured for 24 h in vitro(P<0.01),and could reduce the death of neutrophil NETosis(P<0.01),which was more conducive to promoting the resolution of inflammation,and the role of LXA4 was mediated by ALX receptor.Conclusion:1.LXA4 can protect ARDS rats by affecting the function of peripheral blood neutrophils.2.In the acute phase of inflammation,LXA4 can accelerate the migration of neutrophils to inflammatory sites by promoting the release of NE and MPO of neutrophils and enhance the bactericidal ability of neutrophils.3.NE and MPO play an important role in the process of the formation of NETs in vitro.LXA4 promotes the release of NETs in ARDS rats,which is achieved by affecting the release of NE and MPO.4.During the period of inflammation resolution,LXA4 can accelerate the inflammation resolution by promoting the apoptosis of neutrophils and reducing the death of NETosis.