Effects Of Neutrophil Extracellular Traps On Acute Lung Injury In Neonatal Rats

ObjectiveTo investigate the role of neutrophil extracellular traps(NETs)in neonatal rats with acute lung injury(ALI).Methods30 rats born at 7 days old were randomly divided into normal saline control group,LPS acute lung injury group(ALI group)and ALI+Dnase group,each group has 10 rats.ALI group was intraperitoneally injected with LPS at 20mg/kg,and ALI+Dnase group was intraperitoneally injected with deoxyribonuclease(Dnase)at 5mg/kg after LPS injection.After 6 hours,the rats were anesthetized with chloral hydrate,alveolar lavage fluid(BALF)was collected,and the content of circulatingfree deoxyribonucleic acid(cf-DNA)in BALF was detected by fluorescence microarray.The right lung tissues were fixed in 4%paraformaldehyde,and the morphology and structure of the lung tissues were observed by HE staining.The content of IL-6,IL-8and TNF-αin the lung homogenate was detected by ELISA.Immunofluorescence and Western blot were used to detect the production of citrulline histone 3(Cith 3)and myeloperoxidase(MPO)in lung tissues of rats in each group.Neutrophils were extracted from the heart blood of 7-day-old neonatal rats and divided into control group(LPS group)and LPS+DNase group(LPS+DNase group)with 1×10~5 cells per group.The concentration of 100μmol/L LPS was added into LPS group,LPS+DNase group was added with 5μg/m L of DNase after adding LPS,and the formation of NETs was detected by immunofluorescence technique and Western blot.The neutrophils that had generated NETs were co-cultured with AECII,then the apoptosis of AECII was detected by flow cytometry,and the levels of cf-DNA,IL-6,IL-8,and TNF-αin the supernatant were detected by ELISA.The experimental data were expressed as mean±SEM,and statistical analysis was conducted by one-way ANOVA using SPSS software,P<0.05 represented statistical significance,and Graph Pad Prism 7.0 software was used for plotting.ResultsCompared with the control group,the levels of cf-DNA,Cith 3,MPO,IL-6,IL-8and TNF-αin the ALI group and the ALI+Dnase group were all increased(P<0.05).Compared with the ALI group,the levels of cf-DNA,Cith 3,MPO,IL-6 and TNF-αin the lung tissues of neonatal rats in the ALI+Dnase group were decreased(P<0.05),and the degree of inflammatory infiltration was decreased in the ALI+Dnase group.In vitro test,compared with the control group,the levels of Cith 3 and MPO in the LPS group and the LPS+Dnase group were all increased(P<0.05).Compared with the LPS group,the levels of Cith 3 and MPO in the LPS+Dnsase group were decreased(P<0.05).The apoptosis of AECII was not affected by co-culture with NETs(P>0.05)Compared with the control group,the levels of cf-DNA IL-6,IL-8 and TNF-αin the supernatant of NETs group and NETs+DNase group were increased(P<0.05),the levels of cf-DNA IL-6,IL-8 and TNF-αwere decreased in the NETs+DNase group compared with the NETS group(P<0.05).ConclusionIn neonatal rats with ALI,NETs exists in lung tissue,and NETs level is an important indicator of lung tissue injury.The inhibitor of NETs(DNase)can effectively reduce lung tissue injury,and NETs cannot promote or inhibit AECII apoptosis,but can promote the secretion of various inflammatory mediators.