| BackgroundAcute lung injury(acute lung injury,ALI)is an acute non-cardiogenic pulmonary edema and stubborn hypoxemia of clinical syndrome[1],the critical condition,easy to progress to the acute respiratory distress syndrome,a serious threat to the patient's quality of life and survival,although the critical medical level has been great progress in our country,but for the understanding and treatment of acute lung injury(ALI)still needs further.Its common causes include severe infection,shock,trauma,burns,the large number of blood transfusions,but with the breathing machine assisted ventilation in clinical applications increasingly widespread,lung injury caused by mechanical ventilation(ventilator induced lung injury.VILI)increase gradually and the attention of more scholars.Mechanical ventilation stress damage,caused by the destruction of lung tissue in the membrane structure,the release of inflammatory markers,cause lung injury[2].Studies confirm that renin angiotensin system(reninangiotensin system,RAS)is a local exists in the heart,kidney,brain,lungs and other tissues,including angiotensin?(Ang ?)as the main effector molecule,is by the angiotensin I(angiotensin I.AngI)in angiotensin converting enzyme(angiotensin converting enzyme,ACE)under the action of transformation.The role of the RAS system in acute lung injury has caused wide public concern,studies have confirmed that caused by mechanical ventilation in acute lung injury is closely related to the activation of the RAS system,the application of angiotensin converting enzyme inhibitors or angiotensin ?receptor antagonist,different degrees of acute lung injury relie[3],the study confirms that in the process of acute lung injury(ali)lesions,local renin-angiotensin aldosterone system(RAAS)can cause oxidative stress levels in the organization to add weight target organ damage[4].Studies have confirmed that in the process of the acute lung injury induced by mechanical ventilation,nest protein-1 part in[7][8].Mechanical ventilation in acute lung injury(ali)caused by the process,the lungs whether RAS system through the nest protein-1 also increase oxidative stress levels,causing the inflammatory pathological changes of cells and tissues,it is not clear.Since the 1950s by Palade[5]and Yamada[6],since the first reported in most animals cytoplasmic membrane on the surface of the fine structure-flask sample sag fossa(caveolin)is more and more attention from scholars.The study found that caveolin in cell inside and outside material transportation,not only was a key structure enrichment and signal transduction of cell surface.According to the difference of function and alveolus can be further divided into the structure of the protein caveolin-1(cav-1),of caveolin-2(cav-2)and caveolin-3(cav-3),among them,the most widely in the role and distribution of cav-1,the most comprehensive functions,is the most thorough study.Studies confirm that cav-1 not only participate in the process of acute lung injury lesions,fair can play an important role in it.As one of important signal control platform on the cell membrane surface,fossa and alveolus protein cav-1 in stress lung injury caused by mechanical ventilation in the process of play to the readjustment of the key[8]But for cav-1 specific role in acute lung injury,academia there is still more controversial.The scholar thinks,in the process of mechanical ventilation of cav-1 in high expression in lung tissue can play a significant anti-inflammatory effect[7];But also has the study found that cav-1 can be mediated by the neutrophils inflammation,participate in the occurrence and development of acute lung injury[8].So for cav-1 during acute lung injury play a role of anti-inflammatory and proinflammatory,has not been determined,even has some scholars put forward:cav-1 in acute lung injury,early proinflammatory effects,while in the late injury by inhibiting fibrosis of lung tissue and play a role of the protection of the lungs.NLRP3 inflammatory corpuscle is a combined by a variety of protein complexes,the NLRP3,apoptosis related point sample protein(ASC)and Pro-caspase-1,as the core of NLRP3 inflammatory corpuscle protein NLRP3,related to inflammation of the complex components,such as apoptosis related point sample protein(A6C)and Pro-caspase-1 reg,ulation is of great significance.Studies have found that in acute lung injury,cells after the thorn,increased formation of ROS,activate the NLRP3 inflammatory corpuscle,activating caspase-1,promote pro-IL-1 beta.pro-IL-18 cutting mature,the release of inflammatory factor of lung tissue damage[9].Acute lung injury of acute inflammation,damage,including capillary barrier peqmeability increase,a large number of protein leakage,the release of cytokines.ROS decreased lung compliance,non cardiac pulmonary edema,abnormal gas exchange,cause serious lack of oxyge[23][24][25][26][27][28].This study proposed by searching fossa in lung injury model in mice caused by the change of protein and its related signaling pathways,for the prevention of lung injury caused by mechanical ventilation and treatment to provide new theoretical basis.ObjectiveExcessive use of breathing machine ventilation machine breathing lung injury in mice model was established,and the angiotensin converting enzyme inhibitors ?chloride to intervene with sand,by detecting the lung tissue of mice fossa protein related to oxidative stress protein interaction,assessment of the protein involved in oxidative stress and inflammation in organization related protein expression,discusses chloride sand jotham by caveolin-1 in the breathing machine is the role of lung injury mechanism.Methods1?Experimental animals:36 adult male C57 mice were randomly divided into Control group(Control),pure chlorine temple to medicine group(Losartan,LST),Mechanical ventilation group(Mechanical ventilatioin,MV)and treatment group(MV+ LST)four groups,each group of nine only.Pure chlorine sand temple to the medicine group and treatment group in mice by 10 mg/kg Losartan lavage line 1 week in advance.MV,MV + LST group mechanical ventilation is adopted to establish the model of lung injury in mice,the Control group,the LST group mice don't do processing or a single drug treatment,not breathing machine processing.after the models is established,use the dislocation method to death all the mice,to return alveolar lavage fluid,the lung tissue of mice specimens used for further research.2?Alveolar lavage detection:mice by BCA detection kits to return protein concentration in the alveolar lavage fluid,through the comparison between different groups,the differences in the protein concentration of alveolar lavage judge the differences between groups of acute lung injury in mice.3?Histopathologic evaluation:the preparation of good mice lung tissue specimens of paraffin slice,dewaxing,hydration,and HE staining,Smith scoring method is used to analyse the groups of mice with acute lung injury score,statistical differences between groups of acute lung injury in mice.4?Immunohistochemical staining:preparation of mice lung tissue specimens of paraffin slice,dewaxing,hydratio.antigen repair after processin.such as inactivated endogenous peroxidase,closed the first antibody and nonspecific ant ibody incubation with this second antibody with fluorescent signal,finally dyed with DAPI nuclear and sealing piece.With fluorescent microscope and photogr aphed analysis of experimental results.5?Histologic immunofluorescence double staining:preparation of mice lun g tissue specimens of paraffin slice,dewaxing,hydration,antigen repair after p rocessing,such as inactivated endogenous peroxidase,closed the first antibody and nonspecific antibody incubation with this second antibody with fluorescent signal,finally dyed with DAPI nuclear and sealing piece.With fluorescent mi croscope and photographed analysis of experimental results.6?Western blot:take the left lung tissue preparation tissue homogenate in mice,finish the steps such as centrifugal,determination of concentration and high temperature degeneration,will be mixed with total protein extract of sample buffer to join preparation beforehand good acrylamide gel electrophoresis for protein electrophoresis tank,purpose after protein separation,further transfer film,non-specific antigen and antibody incubation,closed,after the completion of the infrared imaging in detecting fluorescence value target protein and semi-quantitative analysis.Result1?Lung tissue pathological changes and lung injury score:HE staining were observed under optical microscope,the control group and pure chlorine sand temple to medicine group in the bronchial epithelium at various levels and the lung tissue of mice structural integrity,alveolar interval is normal,not seen obvious inflammatory cell infiltration.Mechanical ventilation can be seen in the group of mice lung lots of inflammatory cells infiltration and diffuse pulmonary interstitial edema,alveolar interval obvious thickening,part of the alveolar rupture,alveolar cavity with bloody fluid and inflammatory cells infiltration.Treatment way the visible in the lung of mice inflammatory cell infiltration,thickening of the alveolar interval has a certain degree,but the mechanical ventilation group mice significantly reduce.Smith grading method is used to assess each degree of lung injury in mice,the results show that compared with the control group and pure chlorine dosage group,sand lung injury in mice was significantly increase mechanical ventilation group,treatment group mechanical ventilation group significantly reduce degree of lung injury in mice.2?Alveolar lavage fluid total protein content detection:total protein concentration in groups of mice alveolar lavage fluid,the results show that compared with the control group and pure chlorine sand to medicine group,mechanical ventilation alveolar significantly increased the total protein content in mice,and the treatment group total protein content in mice alveolar lavage group decreased significantly compared with mechanical ventilation.3?Immunofluorescence double dye:groups of mice lung tissue immunofluorescence staining results showed that compared with the control group and pure chlorine dosing of mice with sand,mechanical ventilation group in the lung tissue of mice NOX4 protein(green fluorescent tags)and cav overlap between protein 1(red fluorescent tags)significantly increased,prompting NOX4 protein on the obvious enrichment occurs in the caveolin,and chlorine treatment group in the lung tissue of mice with sand,cav 1 protein fluorescence intensity is abate,and cav 1 NOX4 protein and protein overlapping the mechanical ventilation group decreased significantly in mice,the prompt NOX4 protein within the alveolus enrichment degree is smaller.4?lmmunohistochemical detection It-1 beta content:IL-1 beta protein involved in lung tissue inflammation of mice in different situation expressed in the lung tissue,the results show that mechanical ventilation group in the lung tissue of mice IL-1 beta protein expression was obviously higher,and use of chlorine treatment group in the lung of mice with sand,IL-1 beta expression after mechanical ventilation with mechanical ventilation group decreased significantly in mice.5.Western blot test results:protein imunoblot experiment testing different groups in the lung tissue of mice.NOX4,caveolin-1,IL-1 beta,NLRP3,ASC,pro-caspasell,caspasel-p10 protein expression.The result shows:the caveolin-1 cav 1 group during mechanical ventilation in the lung of mice increased significantly,while the LST after treatment,mechanical ventilation caused by cav 1 protein expression significantly suppressed;About the detection of NOX4 protein involved in oxidative stress,according to the mechanical ventilation group NOX4 protein expression in lung tissue of mice was increased significantly,LST treatment group NOX4 protein expression in lung tissue of mice with mechanical ventilation group decreased significantly in mice;of IL-1 beta protein involved in inflammation and inflammatory corpuscle NLRP3 components,ASC,pro-caspasel,caspasel-p10 test also shows that:mechanical ventilation in mice lung inflammation in related protein expression and inflammatory corpuscle related components significantly increased,while the LST treatment can obviously reduce the mechanical ventilation in the lung tissue of mice caused by inflammmation factors and the expression of inflammatory corpuscle.Conclusion1?In the process of acute lung injury in mice induced by ventilator,the up regulation of caveolin-1(Cav-1)?NOX4?NLRP3 inflammasome are closely related to.2?The effect of acute lung injury induced by ventilator resistance of losartan,and the down regulation of caveolin-1(Cav-1)?NOX4 and NLRP3 inflammasome are closely related to. |
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