The Changes Of NLRP3 Inflammasome And Mitochondria During Ventilator-induced Lung Injury

ObjectiveVentilator induced lung injury(VILI)is a kind of acute lung injury which combines the primary diseases caused by inflammation and injuries of lung tissue during the application of mechanical ventilator.Mechanical factors can destroy the structure and the function of the alveolar membrane barrier through mechanical forces,increase the permeability of epitheliums,also mechanical factors will trigger the inflammatory signaling pathways which induce the release of the inflammation factors leading to lung injury.NLRP3 inflammasome is an important part of innate immune system,NLRP3 protein has critical function that senses harmful stimulations and activates the NLRP3 inflammasome.Activation of the inflammasome transfers the inflammatory factor pro-IL-10 into a mature active form and releases inflammatory factor to extracellular matrix,which causes the inflammation.In the process of the inflammasome activation the reactive oxygen species(ROS)derived from mitochondria plays a key role.The destruction of mitochondria causes a series of consequence such as attenuated generation of ATP,significantly increasing level of ROS which initiates and exacerbates inflammatory reactions.Thus this research aimed to elucidate the interaction between NLRP3 protein and mitochondria during VILI in vivo and in vitro model?MethodIn vivo,thirty SPF C57BL/6 mice,weighing 20-25g,were randomly divided into 3 groups(n=10):the control group(group C),mechanical ventilation group(group MV),mechanical ventilation + TAK-242 group(group MV+T).The mice in group C did not have mechanical ventilation;group MV,group MV+T mice were ventilated witih respiratory frequency for 70/min,tide volume was 15ml/kg,inspiratory-to-expiratory ratio of 1:2,and the inspired oxygen concentration for 21%,the time span is 4h.The mice in group MV+T were pretreated with the TLR4 inhibitorTAK-242(3mg/kg)for 1 h.Lung injury score was recorded after mechanicalventilation.The upper lobe of the right lung was rapidly put into liquid nitrogen freezing to measure the expression of NLRP3 protein,NF?B-P65 and phosphorylation of NF?B-P65 by western blotting;the other part of the right lung was fixed in 4%paraformaldehyde to stain by HE;the left lung was used for collecting the bronchoalveolar lavage fluid to evaluate the level of IL-1?.In vitro,MEL-12 cells were randomly divided into 4 groups:control group(group C),cyclic stretch group(group CS),cyclic stretch+TAK-242 group(group CS+T)and mitochondria respiration chain uncoupler group(group cccp).MLE-12 cells were pretreated with the TLR4 inhibitor TAK-242(luM)1h before cyclic stretch,cccp()were added into culture medium of group cccp for 30 min.Cells in group C were not treated with cyclic stretch.MLE-12 cells were streched with frequency 0.5HZ,amplitude 20%of cyclic stretch for 4h in group CS and group CS+T.After the pretreatment and cyclic stretch,culture mediums were rapidly collected for detecting the level of IL-1?;parts of MLE-12 cells were lysed rapidly on the ice to pick up the total protein to measure the expression of NLRP3 protein NF?B-P65 and phosphorylation of NF?B-P65 by Western Blotting;the rest of MLE-12 cells were used to evaluated the mitochondria membrane potential and the level of ROS were tested by flow cytometry;ResultIn vivo1.HE staining showed that high tidal volume mechanical ventilation would cause alveolar congestion seriously,hemorrhage,and disrupted and thicken alveolar wall were observed,and hyaline membrane formation.After pretreated with TAK-242,alveolar congestion and bleeding reduced,and alveolar wall thicken could alleviated.2.Western blotting suggested that mechanical ventilation led the increasing level of NLRP3 protein and phosphorylation of NF?B-P65,the expressions of NLRP3 protein and phosphorylation of NF?B-P65 in group MV and group MV+T were higher than group C significantly,while the expressions of NLRP3 protein and phosphorylation of NF?B-P65 in group MV+T were lower than group MV.3.ELISA showed the level of IL-1? increased in both group MV and group MV+T while the level of IL-1? of group MV+T decreased compared with group MV.In vitro1.Western blotting suggested compare to group C the expression level of NLRP3 protein and phosphorylation of NF?B-P65 in other 3 groups increased significantly,and the levels of the two proteins decreased after TAK-242 added into the culture medium.2.flow cytometry showed compare to group C mitochondria membrane potential in other 3 groups were reduced and the generation of ROS were increased,in the cells were pretreated with TAK-242,the reduction of mitochondria membrane potential was attenuated and level of ROS was decreased compare to group M.3.ELISA showed the level of IL-1? increased in group CS and group CS+T and group cccp,while the level of IL-1? of group M+T decreased compared with group M.ConclusionMechanical ventilation could increase the level of NLRP3 protein and activate inflammatory reactions and damage the mitochondria,which contributed to the ventilator induced lung injury(VILI).