Necrostatin-1 Ameliorates Neutrophilic Inflammation In Asthma By Suppressing MLKL Phosphorylation To Inhibit NETs Release

BackgroundNeutrophils Extracellular Traps(NETs)are a special form of cell necrosis for Neutrophils.Initially,studies indicated that the main function of neutrophils was antibacterial.Later,studies reported that various stimulating factors such as inflammatory factors,LPS,phorbol myristrate acetate(PMA)and and so on could induce NETs formation.Excessive accumulation of NETs or incomplete clearance of NETs will aggravate the inflammatory response,damage normal tissues,and lead to the occurrence and development of a variety of inflammatory diseases.Current research suggests that neutrophil's releasing NETs play an important role in a variety of autoimmune lung diseases,such as asthma,pulmonary interstitial fibrosis,and acute lung injury.Asthma is a heterogeneous chronic airway inflammation characterized by airway inflammation and reversible airway resistance.Neutrophilic inflammation occurs during asthma exacerbation,and especially,in patients with steroid-refractory asthma,but the underlying mechanisms are poorly understood.Recently,a significant accumulation of neutrophil extracellular traps(NETs)in the airways of neutrophilic asthma has been documented,suggesting that NETs play an important role in the pathogenesis.And NETs maybe a new therapeutic target for neutrophilic asthma.As an inhibitor of programmed necrosis,Nec-1 inhibits programmed necrosis of a variety of cells by allostering RIPK1 kinase.Our previous study indicated that Nec-1 inhibited the releasing of NETs,and it has the potential to treat acute or persistent inflammation-related diseases.Therefore,this study intends to further explore the molecular mechanism of the inhibition of neutrophil's releasing of NETs by nec-1 and whether it can be used to alleviate the inflammation of neutrophilic asthma.To provide new targets for the development of new drugs for the treatment of neutrophil asthma.ObjectiveThere is a large accumulation of NETs in the airway of neutrophil asthma,which is common in hormone-tolerant asthma and severe asthma,and lack effective treatment.The purpose of this study is to investigate the molecular mechanism of inhibiting the formation of NETs by Nec-1 and whether it can be used to relieve the airway inflammation of neutrophilic asthma.MethodsIn vitro,firstly,NETs stimulated by PMA to release neutrophils were isolated and collected.The isolated NETs were co-cultured with human bronchial epithelial cells(16HBEs)to observe the effects of different concentrations of NETs on human bronchial epithelial cells.Secondly,we established an OVA/CFA-induced neutrophilic asthma mouse model to observe whether the level of NETs increased in bronchoalveolar lavage(BAL)in this model,and whether the increasing formation of NETs induced by the stimulant of PMA aggravated airway inflammation.Previous studies have found that the formation of NETs could be inhibited by nec-1 and in vivo experiments we investigated whether inhibiting the formation of NETs by nec-1 could alleviated the airway inflammation of neutrophil asthma.Finally,the molecular mechanism of inhibiting the release of NETs by Nec-1 was confirmed by real-time fluorescence analysis and western-blot experiments.ResultsIn vitro experiments confirmed that NETs directly damaged human bronchial epithelial cells.In vivo experiments found that the level of NETs increased in BALF in a mouse model of OVA/CFA-induced neutrophilic asthma and the increase formation of NETs induced by stimulant PMA aggravated airway inflammation.In vivo experiments we confirmed that the inhibition of neutrophil's releasing of NETs by Nec-1 could alleviated airway inflammation in neutrophilic asthma and reduced the level of NETs,total protein concentrations,myeloperoxidase activity and inflammatory cytokines in BAL.Results of real-time fluorescence analysis and western-blot assay indicated that nec-1 inhibits the release of NETs by inhibiting MLKL phosphorylation and pore formation in cell membrane.ConclusionNETs directly damage human bronchial epithelial cells,and Nec-1 could alleviate the airway inflammation of neutrophilic asthma by inhibiting MLKL phosphorylation and preventing neutrophil's releasing NETs.