The Role Of Extracellular Histones-TWIK2 Mediated Activation Of NLRP3 Inflammasome In Alveolar Macrophages In Sepsis-induced Lung Injury

Objective:Sepsis is one of the most common clinical critical illnesses.Sepsis-induced lung injury is the main cause of death in sepsis patients.Alveolar macrophages play a key role in sepsis-induced lung injury.Extracellular histones is a newly found damage associated molecular patterns(DAMPs).Our previous research found that extracellular histones play an important role in acute lung injury.However,the possible inflammation mechanism of extracellular histones in sepsis-induced lung injury has still not been fully elucidated.The K~+channel protein TWIK2 of alveolar macrophages is up-regulated in the inflammatory response,which can activate the NLRP3 inflammasome and participate in the inflammatory response.Thus,we hypothesized that extracellular histones up-regulate the expression of TWIK2 in alveolar macrophages to mediate potassium ion efflux,thereby activating NLRP3inflammasome,and play a role in mediating inflammatory response in sepsis-induced lung injury.To test this hypothesis,this study intends to explore the role and mechanism of extracellular histones-TWIK2 activates NLRP3 inflammasome of alveolar macrophages and play a role in sepsis-induced lung injury from two levels:in vitro and in vivo.Methods:1.In vitro:Mouse primary alveolar macrophage and alveolar macrophage cell line(MH-S)were taken to add LPS and/or different concentrations of exogenous histones to stimulate the cells.Western Blot was used to detect the dynamic expression of TWIK2 in alveolar macrophages at different stimulation times.ELISA was used to detect the levels of LDH and inflammatory factors in the cell supernatant to explore the optimal concentration and stimulation time.To further examine the effects of LPS+exogenous histones on intracellular potassium levels,NLRP3 and ASC protein expression in alveolar macrophages were determined,and we also explored the effects of extracellular histones induced TWIK2 expression on the activation NLRP3 inflammasome.Then,heparin,quinine,and MCC950 were used to intervene,and the changes of the above indexes were detected.2.In vivo:A mouse model of sepsis-induced lung injury was constructed to verify that extracellular histones induced the activation of NLRP3 by up-regulating the expression of TWIK2,and played a role in promoting inflammation in sepsis-induced lung injury.Construction of mouse CLP model to induce lung injury,after the mice were sacrificed,the lung tissues were taken out for H&E staining to detect the pathological changes of the lungs;ELISA was used to detect the expression levels of extracellular histones,LDH and inflammatory factors in the plasma and BLAF;Western Blot was used to detect the expression of TWIK2,NLRP3 and ASC protein in alveolar macrophages;then intervene with extracellular histones neutralizer heparin tail vein injection to detect changes in the above indicators.Elucidate the role of extracellular histones-TWIK2 activates NLRP3 inflammasome of alveolar macrophages in sepsis-induced lung injury,and explore new targets for the treatment of sepsis-induced lung injury.Results:1.In vitro:It was found that extracellular histones can up-regulate the expression of TWIK2 in alveolar macrophages,promote potassium ions efflux,activate NLRP3inflammasome,and promote the secretion of cellular inflammatory factors.After stimulating cells with LPS and different concentrations of exogenous histones,the expression of TWIK2 in alveolar macrophages was significantly increased,the intracellular K~+level reduced,and the expression of NLRP3 and ASC protein in alveolar macrophages significantly increased.The levels of LDH and inflammatory factors IL-1β,IL-18 and TNF-αin the cell supernatant significantly increased.Interventions with heparin,quinine,and MCC950 could reduce the expression of TWIK2,decrease the expression of NLRP3 and ASC protein,and down-regulate the secretion of cellular inflammatory factors.2.In vivo:The results of cell experiments were further verified by in vivo animal experiments,and it was found that the expression of TWIK2,NLRP3 and ASC proteins in alveolar macrophages of sepsis-induced lung injury mice significantly increased.The levels of extracellular histones and LDH,IL-1β,IL-18and TNF-αin the plasma and BLAF all significantly increased.After intervention with heparin,the lung injury of sepsis mice improved,which increased the survival rate of mice and improved the inflammation in the lungs of sepsis-induced lung injury mice.Conclusions:Extracellular histones can promote potassium ions efflux and activate the NLRP3 inflammasome by up-regulate the expression of TWIK2 protein in alveolar macrophages,which play a role in promoting the inflammatory response in sepsis-induced lung injury.Intervention of the extracellular histones-TWIK2-NLRP3signaling pathway can reduce lung injury,and is expected to provide a new target for clinical treatment of sepsis-induced lung injury.