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The role of microvesicles in ventilator-induced lung injuryAIM: To observe the expression of microvesicles in VILI model mice and explore the possible mechanism of microvesicles involved in pulmonary inflammation.Methods: VILI model was established by mechanical ventilation with tidal volume of 20 ml/kg for 1 h,2 h and 4 h.Lung tissues of mice in each group were stained with H&E to observe the pathological changes of lung tissues after VILI.Lung edema of mice in each group was observed by W/D.Total protein concentration in alveolar lavage fluid was measured by BCA method,lung permeability of VILI mice was observed.The concentration of inflammatory factors such as IL-1beta,IL-6 and TNF-alpha in alveolar lavage fluid was detected by ELISA,and the total number of cells in alveolar lavagefluid was measured by cell counting plate to determine the degree of alveolar inflammation.The expression of Flotillin-2 in lung tissue was detected by Western Blot,and the relationship between the degree of inflammation of VILI and the expression of microvesicles was detected.Microvesicles in alveolar lavage fluid were separated and purified by ultra-high-speed differential centrifugation.The expressions of IL-1beta,IL-6,TNF-alpha,NLRP3 and COX4 in alveolar lavage fluid were detected by ELISA.The active molecular components of microvesicles were identified.Microvesicles were identified by transmission electron microscopy and their morphological characteristics were observed in mechanical ventilated mice and normal mice.Finally,the microvesicles of VILI model mice were isolated and purified and injected into normal mice through trachea to observe the inflammation caused by VILI-derived microvesicles.Result: After mechanical ventilation with 20 ml/kg tidal volume for 1h,2 h and 4 h,there were obvious inflammation changes in lung tissue pathology,destruction of alveolar structure,widening and thickening of pulmonary septum,obvious infiltration of inflammatory cells,and obvious consolidation of lung.At the same time,the concentration of total protein in alveolar lavage fluid increased significantly,alveolar permeability increased significantly,and the expressions of IL-1beta,IL-6 and TNF-alpha in alveolar lavage fluid were significantly elevated.The changes of pulmonary inflammation above were aggravated with the increase of ventilation time.The expression of microvesicles in lung tissue increased significantly in VILI mice,and increased gradually with the increase of ventilation time.Electron transmission microscopy showed that the diameter of microvesicles in mice ventilated with 20 ml/kg tidal volume was larger than that in normal ventilationgroup and spontaneous ventilation group,and the expression of microvesicles was higher,which was strong correlated with ventilation time and reached at the peak at 4 hours of mechanical ventilation.The results of ELISA showed that the contents of IL-1beta and TNF-alpha in the microvesicles of 20 ml/kg tidal ventilation group were significantly higher than those of the spontaneous breathing group and the normal tidal volume group;Among them,the content of inflammatory factors reached the peak at 2 hours of tidal ventilation with20 ml/kg,and the content of IL-1beta and TNF-alpha at 4 hours of ventilation was lower than that at 2 hours,but still higher than that at 1 hour and normal ventilation mice;However,IL-1beta and TNF-alpha in alveolar lavage fluid were reach the peak after 4 hours ventilation.NLRP3 and COX4 were not detected in microvesicles.After the microvesicles of purified from VILI mice,they were injected into normal mice,the pathological changes of lung tissues in mice indicated that the lung showed inflammatory changes,and the concentration of total protein in alveolar lavage fluid increased as well as the inflammatory factors.Conclusion: 20ml/kg tidal volume mechanical ventilation can induce increased release of pulmonary microvesicles,which is strong correlated with the duration of mechanical ventilation.In VILI model mice,microvesicles package IL-1beta and TNF-alpha more than the control mice.Pulmonary inflammation in mice can be induced by VILI-derived microvesicles via injecting into normal mice,suggesting that microvesicles participate in the occurrence and development of VILI by encapsulating IL-1beta and TNF-alpha.Part ? Identify the cellular origin of microvesicles in VILIObjective: To further clarify the cellular origin of inflammatory microvesicles in VILI by flow cytometry.Methods: Mice were divided into three groups by random numbers:spontaneous breathing group,low(normal)tidal volume mechanical ventilation group(7ml/kg)and high tidal volume mechanical ventilation group(20ml/kg)for 4 hours.The pathological changes of lung tissues were observed by H&E staining,the total protein concentration in alveolar lavage fluid was determined by BCA,the inflammatory factors of IL-1beta,IL-6 and TNF-alpha in alveolar lavage fluid were detected by ELISA,and the pulmonary edema was assessed by W/D value.The success of the VILI model is verified by the above methods.After incubating neutrophils(Ly6G+),alveolar epithelial cells(Ep CAM+)and alveolar macrophages(CD11C+/F4/80+)with flow cytometric antibodies,the alveolar lavage fluid of mice in each group was extracted and localized on a flow cytometric machine by 1000-nm microspheres in advance.The detection unit expressing the corresponding antibodies was the cell-derived microcapsules.Immunofluorescence staining(F4/80: alveolar macrophages,Flotillin-2:microvesicles)was used to determine the expression of microvesicles in alveolar macrophages of 20 ml/kg tidal ventilation and spontaneous breathing mice.Result: After 4 hours of high tidal volume mechanical ventilation,the pathological changes of lung tissue were obvious inflammation;W/D results indicated that there was obvious edema in lung tissue;the expression of total protein and inflammatory factors such as IL-1beta,IL-6 and TNF-alpha in alveolar lavage fluid increased significantly.The above results indicate that VILI model can be induced after mechanical ventilation with 20 ml/kg tidal volume for 4 hours.The flow pattern of alveolar lavage fluid showed that the expression of alveolar epithelial cell-derived microvesicles was low in VILI model;although neutrophil-derived microvesicles were highly expressed in VILI,no difference was observed in high ventilation group when compare with normal tidal volume group and spontaneous breathing group;The expression of alveolar macrophage-derived microvesicles was increased in VILI,and the expression of 20ml/kg tidal volume group was significantly higher than that of normal tidal volume group and normal tidal volume group and the spontaneous breathing group.Immunofluorescence staining showed that alveolar macrophages had highly expressed microvesicles after 4 hours of high tidal volume mechanical ventilation.Conclusion: Mechanical ventilation with tidal volume of 20 ml/kg for4 hours can induce pulmonary inflammation.The expression of microvesicles derived from neutrophil is high in lung tissue,but there is no difference in high ventilation groups when compare with low ventilation group and spontaneous group,indicate that neutrophil contributes little to inflammatory microvesicles in VILI;The expression of alveolar epithelial cell-derived microvesicles in VILI model is low;Alveolar macrophage-derived microvesicles play a dominant role in the development of VILI.Part ? The mechanism of alveolar macrophage-derived microvesicles in VILIAIM: In the first and second parts,we clarified the involvement of alveolar macrophage-derived microvesicles in the mediation of VILI.In this part,we explored the regulation of Rho A/Rock signaling pathway on the formation of microvesicles and clarified the molecular mechanism of VILI mediated by alveolar macrophage-derived microvesicles.Methods: Mouse alveolar macrophages were isolated and purified,and the purity was identified by flow cytometry.VILI model of alveolar macrophages in vitro was prepared by using 20% cell stretch strength and cell stretch apparatus.The concentration of IL-1beta,IL-6 and TNF-alpha inflammatory factors in culture supernatant was detected by ELISA to verify the success of VILI model in vitro.Western Blot was used to detect the expression of Rho A,Rock1,Rock2,phosphorylated Limk and microvesicles in alveolar macrophages after 20% stretch stimulation.The relationship between the molecular changes of Rho A/Rock signaling pathway and the expression of microvesicles was observed.Rho A-sh RNA were used to knock down the Rho A signal in alveolar macrophages and then mechanical stretching was performed to observe the expression of microvesicles and the concentration of IL-1beta,IL-6 and TNF-alpha in culture supernatants after Rho A knocking down,and to clarify the regulation of Rho A on microvesicle formation and its effect on inflammation.Rock1-sh RNA were also used to knock down the Rho A/Rock signaling pathway to further explore the regulation of Rho A/Rock signaling pathway on microvesicle formation in VILI model.Results: Flow cytometry showed that the purity of the alveolar macrophages was over 99%,which could be used in subsequent cell stretch experiments.The concentration of IL-1beta,IL-6 and TNF-alpha in the supernatant of alveolar macrophage culture was significantly increased with20% stretch intensity by cell stretch meter,and the inflammation manifestation was obvious.At the same time,the expression of Rho A,Rock 1,Rock 2,phosphorylated Limk and other molecules in alveolar macrophages was significantly up-regulated after 20% cell stretching,and the expression of microvesicles was also significantly increased,and the expression of microvesicles was strongly correlated with the expression of Rho A/Rock signaling pathway molecules.After Rho A knocking down the expression of Rho A,Rock 1 and phosphorylated Limk molecules was down-regulated,and the formation of microvesicles was significantly reduced.Meanwhile IL-1beta,IL-6 and TNF-alpha in culture supernatant were down-regulated and inflammation was improved.Rock1 knocking down have similar effects as Rho A blocking.Conclusion: Alveolar macrophages can induce obvious inflammation after stretching by 20% of cells.At the same time,the expression of Rho A,Rock1,Rock 2 and phosphorylated Limk can be up-regulated and the formation of microvesicles increased.After knocking down Rho A and Rock,the expression of Rock 1 and phosphorylated Limk can be inhibited,the formation of microvesicles can be significantly reduced,the concentration of IL-1beta,IL-6and TNF-alpha in culture supernatant decreases,and inflammation improved greatly.Rho A/Rock signaling pathway regulates alveolar macrophage microvesicle formation in VILI... |
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