The Mechanisms Of NEK7 Regulation Of NLRP3 Inflammasome Activation In Ventilator-induced Lung Injury

Part Ⅰ:Ventilator-induced lung injury is alleviated by inhibiting NLRP3 inflammasome activationBackgroundWith the development and progress of different ventilation modes,mechanical ventilation(MV)is an important life saving means for improving ventilation dysfunction in patients,maintaining alveolar ventilation,reducing carbon dioxide retention,improving the efficiency of pulmonary gas exchange and restoring respiratory muscle function.MV has become an indispensable measure to save the lives of patients who with acute lung injury(ALI)or acute respiratory distress syndrome(ARDS),especially with novel coronavirus infection in severe cases have respiratory failure or apnea.Numerous studies have shown that MV is a double-edged sword,it not only aggravates lung injury such as ALI and ARDS even damages to health,when respiratory parameters settings or with lung function status does not match,eventually lead to the oxygenation dysfunction and pulmonary edema,known as ventilator-induced lung injury(VILI).Despite of a lot of research and exploration on VILI,all kinds of protective lung ventilation measures such as to take out positive end expiratory pressure(PEEP)with low tidal volume as well as recruitment maneuver and permissive hypercapnia.However,due to the complexity and diversity of the pathogenesis of VILI,the complications of VILI still seriously threaten the life of patients.Many studies have confirmed mechanical injury and biotrauma complement each other,are the main cause of VILI.Mechanical injury including barotrauma,volutrauma and atelectrauma,moreover,biotrauma refers to the transformation of mechanical stimulation into biochemical signals and activation of inflammatory cells such as macrophages and neutrophils in the lung through signal transduction pathways,which leads to the release of a large number of cytokines and inflammatory mediators,and finally increases the permeability of alveolar capillaries,leading to pulmonary edema and inflammatory lung injury.Inflammasome activation is an important factor in activating inflammatory pathways that lead to biotrauma.Nod-like receptor protein 3(NLRP3)inflammasome is an important part of innate immunity and participates in immune response and the occurrence of diseases,which is composed of NLRP3,apoptosis-associated speck-like protein(ASC)as well as c-ysteinylasparate specific proteinase 1(caspase-1).Cytokines maturation and secretion such as IL-1β and IL-18 rely on NLRP3 inflammasome activation,but the mechanism of NLRP3 inflammasome activation leading to VILI needs to be further explored.Mechanical force destroys the structure and function of alveolar epithelial cells by activating inflammatory signaling pathways,reducing the content of junction proteins and ultimately leading to the destruction of the integrity of alveolar membranes.Cell junction proteins are composed of adherens and tight junction proteins.Among them,adherens junction proteins consist of E-cadherin,p120-catenin(p120)etc,while tight junction proteins include occludin etc.Mitochondrial structure integrity and normal function are the key to maintain energy metabolism of the body.Decreased in mitochondrial membrane potential can lead to oxidative phosphorylation and energy supply disturbance,which impedes the body’s vital activities.The relationship and mechanism between NLRP3 inflammasome activation,cell junction proteins and mitochondrial oxidative energy supply as well as the mechanism leading to VILI are still unclear,and this study will be further explored.ObjectiveMouse lung epithelial(MLE-12)cells were developed by cyclic stretch and the mice were mechanically ventilated to simulate the VILI model.To explore(1)the effects of cyclic stretch MLE-12 cells at different time on assembly and activation of NLRP3 inflammasome;(2)after silencing NLRP3,the effects of mechanical stimulation on cell junction proteins and mitochondrial membrane potential;(3)the effects and mechanisms of mechanical ventilation on VILI in mice after the low expression of NLRP3.MethodsWe selected the FX-5000 T Flexercell Tension Plus system to cyclic stretch MLE-12 cells for 0,2 or 4 h respectively with 20%stretch amplitude at a frequency of 30 cycles/min(0.5 Hz).Co-immunoprecipitation(CO-IP)was used to detect the combination of NLRP3/ASC/procaspase-1 and immunofluorescence(IF)was used to detect the distribution of ASC and caspase-1.Accordingly,appropriate stretch time was selected for subsequent experiments.MLE-12 cells were transfected with NLRP3 siRNA and mice were injected with a liquid complex of NLRP3 siRNA-lipofectamine 2000 through the fundus venous plexus,which were cyclic stretch or mechanical ventilation for 4 h.Western blot,Co-immunoprecipitation,immunofluorescence,flow cytometry,ELISA,hematoxylin-eosin staining and Wet/dry(W/D)weight ratio were used to assess the silencing efficiency of NLRP3,expression of p120 and occludin,combination of ASC/procaspase-1,distribution of p120 and E-cadherin,changes in mitochondrial membrane potential,content of IL-1β,degree of pulmonary edema and lung injury.ResultsAfter cyclic stretch for 0,2 and 4 h,the combination of NLRP3 inflammasome increased in a time-dependent manner,the distribution of ASC and caspase-1 also increased gradually.After cyclic stretch for 4 h,the expression of NLRP3,binding of ASC/procaspase-1 and secretion of IL-1β were increased,while the expression of p120 and occludin,distribution of p120 and E-cadherin were decreased,meanwhile,mitochondrial membrane potential was also decreased.After low expression of NLRP3 in MLE-12 cells,even though under the same conditions,the increased degree of combination of ASC/procaspase-1 and content of IL-1β were reduced,moreover,the decreased degree of the expression of p120 and occludin,the distribution of p120 and E-cadherin as well as mitochondrial membrane potential were declined.After mechanical ventilation for 4 h,the expression of NLRP3 and the content of IL-1β were increased;the expression of p120 and occludin,the distribution of p120 and E-cadherin were decreased;the lung W/D weight ratio and lung injury score were increased;and the prominent features of the lung tissues in the mechanical ventilation group were the destroyed pulmonary architecture,thickened alveolar septa,alveolar hemorrhage and infiltration of inflammatory cell.But,mice with NLRP3 low expression were mechanically ventilated for 4 h,the increased degree of the expression of NLRP3 and the content of IL-1β were decreased;the decreased degree of the expression and distribution of cell junction proteins were reduced,and the degree of pulmonary edema and inflammatory lung injury were also alleviated.ConclusionsCyclic stretch induced assembly and activation of NLRP3 inflammasome and promoted the secretion of IL-1β.Cyclic stretch resulted in decreased expression of cell junction proteins such as p120,occludin,E-cadherin,and mediated the decrease of mitochondrial membrane potential.After the low expression of NLRP3,the activation degree of NLRP3 inflammasome,the increased degree of IL-1β,the degradation degree of cell junction proteins and the decreased degree of mitochondrial membrane potential caused by cyclic stretch were all reduced.After the low expression of NLRP3,the degree of pulmonary edema and inflammatory lung injury induced by mechanical ventilation was alleviated.NLRP3 is the key to NLRP3 inflammasome assembly and activation,and VILI is alleviated after interfering NLRP3 inflammasome activation by inhibiting cytokines release,the degradation of cell junction proteins and relieving mitochondrial dysfunction.Part Ⅱ:The mechanisms of NEK7 mediated assembly and activation of NLRP3 inflammasome downstream of potassium efflux in ventilator-induced lung injuryBackgroundMechanical ventilation is an indispensable measure to maintain ventilation and oxygenation in general anesthesia and critical patients,but it also brings certain harm to save lives,which is called VILI.Mechanical injury such as excessive ventilation pressure,high tidal volume and collapsing atelectasis damage the structure of pulmonary parenchyma and interstitial,impair the integrity of alveolar membrane,eventually present with pulmonary edema and oxygenation dysfunction.The exploration and optimization of a large number of ventilation modes make the prevention and treatment of mechanical injury progress.Studies have proved that mechanical stimulation induces the recruitment of inflammatory cells,the release of cytokines and inflammatory mediators,while abnormal immune regulation and persistent inflammatory damage lead to pulmonary inflammation and even systemic inflammation.However,due to the complexity of inflammatory signaling pathways and other characteristics,inflammatory lung injury is still not effectively prevented at present.Moreover,hypoxemia and multiple organ dysfunction syndrome(MODS)caused by the release of inflammatory mediators and infiltration of inflammatory cells during the process of VILI have attracted more and more attention,which have also become hot and difficult points in the research field of VILI.Previous studies have found mechanical ventilation can increase the level of IL-1β,IL-18 in mice bronchoalveolar lavage fluid and plasma.Caspase-1 can cut precursor IL-1β,IL-18 into mature and promote them release and NLRP3 inflammasome activation is the key to the above steps.So what is the upstream mechanism of regulating NLRP3 inflammasome activation under mechanical stimulation?It was found that NIMA-related kinase 7(NEK7),which is involved in mitosis,is the key protein for assembly and activation of NLRP3 inflammasome.Meanwhile,intracellular potassium efflux can promote activation of NLRP3 inflammasome.If the relationship between potassium efflux and NEK7,and the mechanisms of them in activation of NLRP3 inflammasome can be clarified,it will play an important role in the mitigation and prevention of VILI.ObjectiveAfter interfering potassium efflux or silencing NEK7,MLE-12 cells were subjected to cyclic stretch and a mouse VILI model was established.To study(1)the effect of mechanical force on potassium efflux,the combination of NEK7/NLRP3 and NLRP3 inflammasome activation;(2)the regulatory effect of glibenclamide(glb)on the combination of NEK7/NLRP3 and NLRP3 inflammasome activation after blocking potassium efflux;(3)the mechanism of NEK7 regulation of NLRP3 inflammasome activation;(4)the mechanism of blocking potassium efflux and inhibiting the combination of NEK7/NLRP3 to alleviate VILI.MethodsFX-5000 T Flexercell Tension Plus system was used to cyclic stretch MLE-12 cells for 0,2 or 4 h respectively with 20%stretch amplitude at a frequency of 30 cycles/min(0.5 Hz).Atomic absorption spectroscopy(AAS)was used to detect intracellular potassium concentration while assessing the extent of potassium efflux.CO-IP was used to detect the binding of NEK7/NLRP3,NLRP3/ASC/procaspase-1,and Western blot,immunofluorescence were used to detect the expression and distribution of NEK7 and NLRP3.After potassium efflux was blocked by glibenclamide,MLE-12 cells were pretreated with cyclic stretch for 4 h.The combination of NEK7/NLRP3 and NLRP3/ASC/procaspase-1 was detected by CO-IP,the expression of NLRP3 was detected by Western blot,and the distribution of caspase-1,NLRP3 and IL-1β were detected by immunofluorescence.NEK7 siRNA was used to silence NEK7 in MLE-12 cells,which were subjected to cyclic stretch for 4 h,Western blot was used to detect silencing efficiency.The binding of NLRP3/ASC/procaspase-1 was detected by CO-IP and the distribution of IL-1β was detected by immunofluorescence.C57BL/6 mice were intraperitoneally injected with glibenclamide or oridonin(Ori)and dimethyl sulfoxide(DMSO)at corresponding concentrations,which were randomly divided into mechanical ventilation group and sham group.Mice in mechanical ventilation group were underwent tracheostomy with a catheter and mechanical ventilation for 4 h,the tidal volume was 28 ml/kg,a frequency was 60 breaths/min and end-expiratory pressure was 0 cm H2O,but the sham group mice were intubated without mechanical ventilation.C57BL/6 mice without pretreatment were mechanically ventilated for 0,2 and 4 h respectively.(1)CO-IP was used to detect the binding of NEK7/NLRP3 and ASC/procaspase-1 after mechanical ventilation for 0,2 and 4 h.(2)Mice pretreated with glibenclamide were mechanically ventilated for 4 h,the binding of NEK7/NLRP3 and ASC/procaspase-1 was assessed by CO-IP,the expression of NLRP3 was selected by Western blot,immunofluorescence was used to detect the distribution of caspase-1,NLRP3 and IL-1β,lung W/D ratio was selected to evaluate the degree of pulmonary edema,H-E staining as well as lung injury score were used to detect the degree of lung injury and Masson staining was used to assess the degree of pulmonary fibrosis.(3)C57BL/6 mice pretreated with oridonin were mechanically ventilated for 4 h,CO-IP was used to detect the binding of NEK7/NLRP3 and NLRP3/ASC/procaspase-1,immunofluorescence was used to detect the distribution of IL-1β,lung W/D ratio was used to evaluate the degree of pulmonary edema,the degree of lung injury and pulmonary fibrosis was evaluated by H-E staining,lung injury score and Masson staining.ResultsAfter cyclic stretch for 0,2,4 h,intracellular potassium concentration decreased in a time-dependent manner,that is,potassium efflux increased in a time-dependent manner.Compared with the control group,the combination of NEK7/NLRP3 and NLRP3/ASC/procaspase-1 were increased after cyclic stretch for 2 h,but the combination degree of them was more significant after cyclic stretch for 4 h.The combination of NEK7/NLRP3 and NLRP3/ASC/procaspase-1,the expression of NLRP3,and the distribution of caspase-1,NLRP3 and IL-1β were all increased after cyclic stretch for 4 h,while the above increases were reduced after MLE-12 cells pretreatment with glibenclamide and cyclic stretch for 4 h.After low expression of NEK7 in MLE-12 cells,which were subjected to cyclic stretch for 4 h,the results showed that the increased binding of NLRP3/ASC/procaspase-1 and the distribution of IL-1β were decreased.The binding of NEK7/NLRP3 and ASC/procaspase-1 showed increased in a ventilation time-dependent manner.Glibenclamide pretreatment group underwent mechanical ventilation for 4 h showed that the increased binding degree of NEK7/NLRP3,ASC/procaspase-1,expression level of NLRP3,distribution of caspase-1,NLRP3 and IL-1β,the level of W/D ratio and lung injury score were all reduced;moreover,the changes of inflammatory lung injury and pulmonary fibrosis were also alleviatived.The increased binding degree of NEK7/NLRP3,NLRP3/ASC/procaspase-1,the increased distribution of IL-1β in lung tissues were reduced,the increased W/D ratio and lung injury score were decreased,and the pathological changes of lung tissues and pulmonary fibrosis were alleviated in oridonin pretreatment group underwent mechanical ventilation for 4 h.ConclusionsMechanical force promoted the binding of NEK7/NLRP3 downstream of potassium efflux,thus accelerating assembly and activation of NLRP3 inflammasome.NEK7 is an essential mediator of NLRP3 inflammasome activation.Low expression of NEK7 inhibited activation of NLRP3 inflammasome induced by mechanical force.Glibenclamide blocked the binding of NEK7/NLRP3,then inhibited activation of NLRP3 inflammasome to play an anti-inflammatory effect by interfering potassium efflux and alleviating VILI in mice.Oridonin played an anti-inflammatory role by blocking the combination of NEK7/NLRP3 and inhibiting NLRP3 inflammasome activation,thereby reducing the degree of pulmonary edema,inflammatory lung injury and pulmonary fibrosis.