An Experimental Study Of Disulfiram On The Treatment Of Acute Lung Injury By Inhibiting The Release Of NETs

Acute lung injury(ALI)is an acute pulmonary disease with non-cardiac dyspnea,hypoxemia as the clinical manifestations,and diffuse alveolar injury as the main pathological features.The therapeutic focus is on ensuring ventilation,fluid management,prevention or treatment of organ failure and management of various complications.However,the clinical treatment has limited efficacy and the mortality rate is high.It is reported that Neutrophil extracellular traps(NETs)have an important role in initiating inflammatory storm and mediating host tissue and organ injury[23-28,30,43],which will be a new therapeutic direction for intervening in the production of NETs in the occurrence and development of acute lung injury.ObjectiveAcute lung injury was treated by inhibiting the release of NETs with the GSDMD inhibitor disulfiram(Disulfiram).This topic is to investigate whether disulfiram can inhibit the release of neutrophil NETs,and explore the role of NETs in the development of acute lung injury,to provide new targets for clinical treatment of acute lung injury.MethodsThis project was verified by both in vivo and in vitro experiments.In vitro experiment:neutrophils isolated from the peripheral blood of healthy volunteers were seeded in cell plates and randomly divided into Control group(negative control),PMA group(positive control),and PMA+Disulfiram group(treatment group).Quantitative immunofluorescence was used to detect the production content of NETs in each group and the direct observation of NETs morphology,the amount of NETs produced in different groups,and the number of viable cells.In vivo experiment:In vivo experiments:the total cell number,alveolar lavage fluid and peripheral serum inflammatory factors(IL-1β,TNF-α)in the ALI+Disulfiram group were lower than those in the ALI group;In ALI+ Disulfiram group,lung edema and protein exudation were significantly less than in ALI group;Lung histopathological sections suggested that the ALI mice had massive inflammatory cell infiltration,pulmonary interstitial edema,and scattered red blood cells in the alveoli;However,the lung histopathological sections of ALI+Disulfiram mice indicated that lung interstitial edema and inflammatory cell infiltration were reduced compared with ALI mice;The content of dsDNA and NETs in the supernatant of alveolar lavage fluid in the ALI+ Disulfiram group was significantly lower than that in the ALI group.ResultsIn vitro experiment:the immunofluorescence intensity of the PMA group determined by microplate reader was significantly higher compared with that of the Control group,and a large number of filaments were formed in the PMA group under the immuno-microscope,indicating that PMA can effectively stimulate neutrophils to produce NETs.The immunofluorescence intensity of the Disulfiram group(disulfiram single agent)was not different from the Control group,and no filformation was observed in the Disulfiram group.The immunofluorescence intensity of PMA+Disulfiram group determined by microplate reader was significantly lower compared with PMA group,and the filaments of PMA+Disulfiram group were significantly lower compared with PMA group by immune microscope,indicating that disulfiram could effectively inhibit the formation of neutrophil NETs stimulated by PMA.In vivo experiments:the total cell number,alveolar lavage fluid and peripheral serum inflammatory factors(IL-1β,TNF-α)in the ALI+Disulfiram group were lower than those in the ALI group;ALI+Disulfiram group were significantly less edema and protein exudation than ALI group;The lung histopathological results suggested that the mice in ALI group had numerous inflammatory cells,pulmonary interstitial edema,and scattered red blood cells in the alveoli;Pulmonary interstitial edema,degree of inflammatory cell infiltration in ALI+Disulfiram group were reduced compared with mice in ALI group;The content of dsDNA and NETs in the supernatant of alveolar lavage fluid in the ALI+Disulfiram group was significantly lower than that in the ALI group.ConclusionDisulfiram can inhibit the release of neutrophil NETs,through which it effectively reduces the condition of LPS-induced ALI mice,and the mechanism may be caused by the inhibition of GSDMD perforation in the neutrophil membrane.