| BackgroundClinical anesthesia maintenance and respiratory failure caused by shock,infection and trauma,as well as oxygenation disorders caused by aging population and air haze,make ventilator treatment become more and more common.Mechanical ventilation has become an essential therapy to save lives in clinical practice.However,improper ventilation parameters and modes can easily cause damage to the airway and pulmonary alveoli,and patients often suffer from aggravation,decreased oxygenation and severe impairment of lung function,which is known as ventilator-induced lung injury(VILI).Once VILI occurs,it is easy to induce systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS),and the mortality rate is very high.VILI has caused a huge economic and social burden,and the prevention and treatment of VILI has become the focus of patients with clinical mechanical ventilation.The pathogenesis of VILI has not been clearly defined,but the common characteristics and primary manifestations of lung injury induced by any cause are pulmonary edema.The main pathophysiological changes of pulmonary edema are increased alveolar membrane permeability,accumulation of fluid in alveolar and interstitial tissues,infiltration of inflammatory factors and inflammatory cells,and hypoxemia.The formation of pulmonary edema will affect the blood and gas exchange and then it will endanger the functions of organs and life safety.Therefore,prevention of pulmonary edema is a key step to avoid the occurrence of VILI.Thus,the prevention of pulmonary oedema is the key to avoiding the occurrence of VILI,and the early prevention of pulmonary oedema can inhibit or prevent the subsequent series of reactions and damageIn VILI,mechanical force,inflammation and hypoxia result in the destruction of alveolar membrane integrity,increase of intercellular space and abnormal pulmonary water transport.In clinical ventilator treatment,the mechanical force first directly acts on alveolar epithelial cells and trachea,followed by vascular endothelial cells and other alveolar wall tissues.It has been reported and our previous studies have shown that as a part subjected to mechanical forces directly,the integrity of the membrane of the pulmonary epithelium,the state of intercellular connections and the shielding and filtering functions of alveolar membrane play a key role in the permeability of the alveolar membrane,and adequate energy support is needed to maintain the integrity and normal function of the cell structure.As the main place of energy supply,mitochondria participate in a number of life activities in cells and play an important role in maintaining the homeostasis of cells.The normal function of mitochondrial energy supply is a prerequisite for the orderly progress of cell activities.The damage of mitochondria caused by various causes can lead to the production barrier,which causes the abnormal functions of cells and the structural and functional changes of tissues and organs.However,whether mitochondria are damaged during VILI,the mechanism of this process and how to prevent this damage still need to be further explored.Previous studies have reported that the NLRP3 inflammasome is involved in inflammation and VILI and that NLRP3 is closely associated with mitochondria.12-14 In response to external changes,such as electricity,LPS or other stimuli,NLRP3 interacts with pro-caspase-1 through ASC,which leads to the activation of caspase-1.Activated caspase-1 promotes the cleavage and maturation of pro-inflammatory cytokines in the cytoplasm(pro-IL-1?,pro-IL-18 and IL-33).Pro-IL-1? is cleaved by caspase-1,and then,mature IL-1? is released.Whether the activation of NLRP3 inflammasomes can directly affect mitochondrial structure and function during VILI is not clearly known.p120 catenin(p120,p120-catenin)is an important protein for cell adhesion junctions,which mainly distributed in vascular endothelial cells,lung epithelial cells and fibroblasts.Previous studies have shown that p120 can stabilize the position of E-cadherin on the cell membrane,inhibit the degradation of occludin and E-cadherin endocytosis,enhance the firmness of intracellular cytoskeleton proteins,thereby preventing increased pulmonary vascular permeability and reducing the incidence of pulmonary edema.Meanwhile,in VILI,p120 inhibited RhoA activity by binding to RhoA,and then inhibited the binding of TLR4 to MyD88,which can effectively block the TLR4 signaling pathway and participate in the body's anti-inflammatory response.Previous studies have focused on the signal transduction mechanism of VILI and the release and accumulation of inflammatory factors.In VILI,is mitochondrial structure or functions that constitutes the alveolar membrane abnormal or damaged?If mitochondria are abnormal,by what mechanism?What are the effects on lung injury?Can inhibition of mitochondrial damage or repair of its function reverse or reduce lung injury pulmonary edema?Changes of NLRP3 inflammatory proteins in VILI and the mechanism of interaction between NLRP3 and mitochondria remain unknown.As a key component of cell adhesion junction,p120 plays the role of a set point.The mechanism by which p120 regulates NLRP3 and its relationship with mitochondria to affect lung injury and pulmonary edema is still unclear.We considered whether the protective effect of p120 on lung injury could not only stabilize membrane permeability and intracellular cytoskeletal protein,but also inhibit the formation of NLRP3 inflammaosomes by inhibiting the signal transduction pathways of TLR4 and ROS,and further inhibit the destruction of NLRP3 on mitochondrial structure and function,thus protecting lung.In this regard,we propose:1.Whether changes in NLRP3 inflammatory protein during mechanical stretch can directly affect mitochondria and induce lung injury?2.Can p120 negatively regulate NLRP3 and protect mitochondria during mechanical stretch?3.Since p120 is a set point protein,can we regulate the expression of p120 to inhibit NLRP3 and further inhibit the activation of NLRP3 inflammasomes,and in order to reduce mitochondrial damage and reduce VILI,and provide a new biological transformation idea for the prevention and treatment of clinical VILI?4.If the above changes can be established,what are their changes,effects and significance in the process of VILI and pulmonary edema in vivo?Therefore,the relationship among p120,NLRP3 and mitochondria needs to be further explored.In this study,in vivo and in vitro VILI models were used to explore the protective effect and mechanism of p120 on mitochondria.We propose a hypothesis:in VILI,p120 can inhibit the activation of NLRP3 by inhibiting the TLR4 signaling pathway and ROS production in oxidative stress,and further reduce the damage to mitochondrial structure and function,and thus prevent the formation of lung injury and pulmonary edema.Meanwhile,p120 not only has a protective effect on mitochondrial function,but also inhibits systemic inflammatory response by inhibiting the active inflammatory factor IL-1? released after activation of NLRP3 inflammomes,which has a broad sense of organ protection.ObjectivesIn this project,two models of cell cyclic stretching and animal mechanical ventilation were established.1.The changes of mitochondria and NLRP3 in the VILI process and their relationship were discussed.2.The negative regulation of p120 on NLRP3 and the possible mechanisms of mitochondrial protection were revealed,so as to provide new ideas for basic research on VILI.3.From the perspective of inflammasomes and mitochondrial productivity,cell junction protein p120 and mitochondria which were the direct action and effective intervention targets in VILI were explained at the source of lung injury,so as to provide new clinical targets for the prevention and treatment of VILI.MethodsThis project used the alveolar epithelial cells,mechanically ventilated lung injury model mice and knockout mice as the research object.This project used specific inhibitor treatment,siRNA transfection technique,western blot,enzyme-linked immunosorbent(ELISA),flow cytometry,electron microscopy,hematoxylin eosin(HE)and evans blue staining as experiment technology.TLR4 signaling pathway,ROS activation,NLRP3 inflammatory protein and their activation products,mitochondrial structural and functional changes,lung injury,and the degree of pulmonary edema were detected to provide a new theoretical basis for the prevention and treatment of VILI.Part 1 The effects of NLRP3 on mitochondria and ROS during cyclic stretchingMLE-12,monolayer-grown mouse lung epithelial cell lines,were cultured in 6-well cell culture plates and BioFlex plates.In this study,Flexcell-5000T Tension System was used to stretch MLE-12 to simulate ventilator ventilation in clinical practice.Cell cyclic stretching parameters:20%cyclic stretching,0.5HZ,a stretch-to-relaxation ratio of 1:1,stretch time 4 hours.In the MCC950 treatment group,NLRP3 inhibitor MCC950 was added to pretreat MLE-12 cells for 1 hour before cyclic stretching.Random grouping:MLE-12 cells were divided into C group(control group),CS group(cyclic stretching group),D group(treated with DMSO for 1 hour),and CS+M group(pre-treated with MCC950 for 1 hours before cyclic stretching).1.JC-1 probe was loaded into cells with fluorescent probe,and the mitochondrial membrane potential change was detected by flow cytometry,and the change was represented by PE/FITC value.2.Cell proteins were extracted from RIPA lysate and the expression levels of NLRP3 and catenin in each group were detected by Western Blot.3.DCFH-DA probe was loaded into the cells,and the level of ROS in each group was detected by flow cytometry.The change of FITC value was used to represent the change of ROS in the cellsPart 2 Effects and mechanisms of p120 on NLRP3 and mitochondria during cyclic stretchingMLE-12,monolayer-grown mouse lung epithelial cell lines,were cultured in 6-well cell culture plates and BioFlex plates.In this study,Flexcell-5000T Tension System was used to stretch MLE-12 to simulate ventilator ventilation in clinical practice.Cell cyclic stretching parameters:20%cyclic stretching,0.5HZ,a stretch-to-relaxation ratio of 1:1,stretch time 4 hours.MLE-12 cells were transfected with Sc siRNA and p120 siRNA to downregulate the expression of p120 gene.The transfection reagent was Lipofectamine 2000.The cells were transfected for 6 hours and then changed into fresh cell culture medium Then the cells in each group were incubated at 37? and 5%CO2 for 48 hours before the subsequent experiments.Random grouping:MLE-12 cells were divided into 1.Sc siRNA group,lOnM p120 siRNA group,30nM p120 siRNA group,and 50nM p120 siRNA group.2.Sc siRNA group,Sc siRNA+CS(cyclic stretching)group,p120 siRNA group,p120 siRNA+CS(cyclic stretching)group.1.MLE-12 cells were transfected with p120 siRNA,and the transfection concentration gradient was set to determine the optimal transfection concentration.2.After the cyclic stretching,RIPA lysate was added to extract MLE-12 cell proteins,and Western Blot was used to detect the expressions of NLRP3,p120,?-catenin,TLR4,PhosphoNF-?B?NF-?B and ICAM1 in each group.3.After the treatment in each group,IL-1? in cell culture supernatant were quantitatively determined by mouse ELISA kit.4.After the treatment in each group,the binding degree and expression distribution of NLRP3 and Caspase-1 in different treatment groups were observed under immunofluorescence confocal microscopy.5.After treatment in each group,the DCFH-DA probe was loaded into cells,and the level of reactive oxygen species(ROS)in each group was detected by flow cytometry.The change of FITC value was used to represent the change of ROS.6.After the cyclic stretching,ultrathin sections were prepared,and the changes of mitochondria and cell internal structure were observed under transmission electron microscope.7.After treatment in each group,the JC-1 probe was loaded into cells,and the mitochondrial membrane potential changes were detected by flow cytometry and expressed by PE/FITC values.Part 3 Effects and mechanisms of p120 on NLRP3 and mitochondria during pulmonary edema induced by mechanical ventilation in miceThe p120 siRNA-liposome mixture was injected into the retinal vein plexus of C57BL/6 mice,and most of the p120 genes in the mice were removed.Then the mice were mechanically ventilated at high tide volume.The mice were pretreated with NLRP3 inhibitor MCC950(4 mg/kg)intraperitoneally 1 hour before ventilation.Parameters of mechanical ventilation of mice:tidal volume was 28ml/kg,ventilation frequency was 60 times/min,ventilation time was 4h,I/E ratio of 1:2.Experimental group:1.C57BL/6 mice were divided into control group,mechanical ventilation group,DMSO treatment group,and MCC950 treatment+mechanical ventilation group.2.C57BL/6 mice were divided into control group,mechanical ventilation group,p120 knockout group,and p120 knockout+mechanical ventilation group.Control group:the mice were not mechanically ventilated.Mechanical ventilation group:the mice were given high tidal volume mechanical ventilation according to the above parameters.DMSO treatment group:I hour before mechanical ventilation,mice were intraperitoneally injected with DMSOMCC950 treatment+mechanical ventilation group:1 hour before mechanical ventilation,mice were intraperitoneally injected with MCC950.Then the mice were given high tidal volume mechanical ventilation according to the above parametersp120 knockout group:300ul p120 siRNA-liposome mixture(7.5nmol siRNA))was injected into the retinal vein plexus of mice for 3 times in total,and the p120 knockout efficiency of the lung tissues of mice was detected by Western Blot.p120 knockout+mechanical ventilation group:300ul p120 siRNA-liposome mixture(7.5nmol siRNA)was injected into the retinal vein plexus of mice for 3 times in total.Mechanical ventilation was conducted and the parameters were set as above.1.Lung tissues of C57BL/6 mice treated in each group were determined by Western Blot for the expressions of NLRP3,p120,?-catenin,TLR4,PhosphoNF-?B?NF-?B and ICAMI in each group.2.After the treatment in each group,IL-1? and IL-6 in bronchoalveolar lavage solution were quantitatively determined by mouse ELISA kit3.HE staining was used to observe the pathological changes of lung tissue.4.Lung tissue was taken to weigh the Wet/Dry weight(W/D)of lung tissue.5.Mitochondria of lung tissue were extracted and mitochondrial membrane potential was detected by flow cytometry,which was expressed by PE/FITC6.Changes in lung permeability after lung injury were detected by Evans blue staining.ResultsPart 1 The effects of NLRP3 on mitochondria and ROS during cyclic stretching1.Effects of NLRP3 on mitochondria during cyclic stretching.Mitochondrial membrane potential showed that compared with the control group,the mitochondrial membrane potential decreased significantly in the cyclic stretching group,indicating mitochondrial damage.When cyclic stretching was performance after treatment with NLRP3 inhibitor,mitochondrial membrane potential increased and mitochondrial damage improved(P<0.05).2.Effects of cyclic stretching on NLRP3.Western Blot showed that the expression of NLRP3 increased after 4 hours of 20%cyclic stretching.When cyclic stretching was performance after treatment with NLRP3 inhibitor,the expression of NLRP3 was decreased compared with the cyclic stretching group(P<0.05).3.Effects of NLRP3 on ROS during cyclic stretching.The detection of intracellular reactive oxygen species showed that compared with the control group,ROS increased significantly in cyclic stretching group.When cyclic stretching was performance after treatment with NLRP3 inhibitor,intracellular ROS decreased(P<0.05).This result indicated that cyclic stretching could induce mitochondrial damage and the NLRP3 inhibitor,MCC950,rescued cyclic stretching-induced mitochondrial damage.Part 2 Effects and mechanisms of p120 on NLRP3 and mitochondria during cyclic stretching1.Effects of p120 on NLRP3 during cyclic stretching.Western Blot showed that with the increase of p120 siRNA transfection concentration,NLRP3 showed a dose-dependent increase in a certain range.Compared with the control group,the expression of NLRP3 increased in the p120 siRNA group and the cyclic stretching group,and this trend was more obvious in the p120 siRNA group after 4 hours of 20%cyclic stretching.Conversely,p120 expression was decreased in the p120 siRNA group compared with the C group,and this effect was enhanced in the p120 siRNA group after 4 hours of 20%cyclic stretching.In addition to the activation of NLRP3,ELISA showed that a product of the activated NLRP3 inflammasome,IL-1?,increased as NLRP3 increased.Confocal immunofluorescence showed that the trend of NLRP3 and Caspase-1 was the same as that of Western Blot(P<0.05).2.Mechanism of p120 regulating NLRP3 during cyclic stretching.(1)The regulatory effect of p120 on TLR4 pathway.The expression levels of TLR4,phospho-NF-?B,NF-?B and ICAM1 were all increased in the p120 siRNA group and CS group compared with the C group,while they were enhanced in the p120 siRNA group after 4 hours of 20%cyclic stretching(P<0.05).(2)Effects of p120 on ROS.Flow cytometry detection of intracellular ROS showed that ROS production was increased in the p120 siRNA group and CS group compared with the C group,and it was further enhanced in the p120 siRNA group after 4 hours of 20%cyclic stretching(P<0.05)3.Effects of p120 on mitochondria during cyclic stretching.Electron microscopy showed that,compared with the C group,the intracellular mitochondria in the p120 siRNA+CS group showed more degenerative signs than those in the CS group,such as edema of mitochondria,an increase in vacuolation.Intracellular mitochondrial membrane potential was detected by flow cytometry,and it reflected early functional changes in mitochondria.We found that MMP was decreased in the CS group and p120 siRNA group compared with the C group,while the MMP of the p120 siRNA+CS group decreased more notably than that of the CS and p120 siRNA groups(P<0.05)p120 prevented the activation of NLRP3 after cyclic stretching.p120 regulated NLRP3 by inhibiting the TLR4 pathway and ROS activation after cyclic stretching.p120 played a vital role in protecting mitochondrial structures and functions after cyclic stretching.Part 3 Effects and mechanisms of p120 on NLRP3 and mitochondria during pulmonary edema induced by mechanical ventilation in mice1.Effects of mechanical ventilation on NLRP3 and lung in mice.Western Blot shows:NLRP3 expression was increased in the MV group compared with the C group,whereas this effect was reversed in the MCC950 pre-treatment group.HE staining and Wet/Dry ratio indicated that lung damage ang edema were aggravated in the MV group,whereas this effect was reversed in the MCC950 pre-treatment group(P<0.05).2.Effects of p120 on NLRP3 during mechanical ventilation.Western Blot showed that the expression of NLRP3 increased in the p120 knockout group and the mechanical ventilation group when compared with the control group,and this trend was more obvious in the p120 p120 knockout group after 4 hours of 20%cyclic stretching.Conversely,p120 expression was decreased in the p120 knockout group compared with the C group,and this effect was enhanced in the p120 knockout group after 4 hours of 20%mechanical ventilation(P<0.05).3.Mechanism of p120 regulating NLRP3 during mechanical ventilation.Western blot analysis revealed that the expression levels of TLR4,phospho-NF-?B,NF-?B and ICAM1 were all increased in the p120 knockout group and mechanical ventilation group compared with the C group,while they were enhanced in the p120 knockout group after 4 hours of 20%mechanical ventilation(P<0.05).4.Effects of p120 on mitochondria during mechanical ventilation.Intracellular mitochondrial membrane potential was detected by flow cytometry.We found that MMP was decreased in mechanical ventilation group and p120 knockout group compared with the C group,while the decrease was enhanced in the p120 knockout group after 4 hours of 20%mechanical ventilation(P<0.05)5.Effects of p120 on pulmonary edema during mechanical ventilation in mice.Evans blue staining,HE staining and Wet/Dry ratio indicated that lung damage ang edema were aggravated in the mechanical ventilation group and p120 knockout group compared with the C group,while the damage was enhanced in the p120 knockout group after 4 hours of 20%mechanical ventilation.Whereas this effect was reversed in the MCC950 pre-treatment group.ELISA showed that,compared with the control group,the inflammatory factor IL-6 and the activated product IL-1? of NLRP3 inflammaomes increased in the p120 knockout group and the mechanical ventilation group,and the increase was more significant after the mechanical ventilation of the p120 knockout group(P<0.05).p120 regulates NLRP3 by inhibiting the activation of TLR4 signaling pathway and ROS activation,thus achieving the protection of mitochondria.ConclusionsIn VILI,mitochondria interact with NLRP3 inflammatory protein,and cyclic stretching activates NLRP3 and leads to mitochondrial damage,while inhibition of NLRP3 can reduce mitochondrial damage caused by cyclic stretching.p120 inhibits the damage of NLRP3 to mitochondria by inhibiting TLR4 pathway and ROS activation,and plays a protective role in lung.Mitochondria and NLRP3 inflammasomes are important targets for the prevention and treatment of VILINew argumentsThis project focused on the key aspects of pulmonary edema:increased alveolar membrane permeability,activation of NLRP3 inflammasomes,and mitochondrial damage in vivo and in vitro.It was more clinically valuable to directly cut off the root of lung injury and provided the possibility of translational medicine than previous studies,providing innovative and practical ideas for clinical intervention and treatmentNew scientific thinking and interventions:From the perspective of inflammasome and mitochondria,in vivo and in vitro experiments are discussed respectively.This project proposed a mechanism study on the regulation of"p120-NLRP3 inflammasome-mitochondria-alveolar membrane permeability-VILI pulmonary edema" with mitochondria and inflammaosome as the core.The project provided new targets for VILI basic research and clinical prevention and treatment of VILI.At the same time,it found a new way for the development and application of p120 biological protein products,inflammasome inhibition and mitochondrial protection drugs. |
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