ABSTRACT
Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.
ABSTRACT
PURPOSE: The aim of this study was to evaluate the effect of inhaled nitric oxide(iNO) on gas exchange, hemodynamics and pulmonary inflammation in newborn piglets with E. coli induced septic lung. METHODS: Twenty three instrumented and ventilated piglets were randomized into three groups: CON(n=6), PCON(n=9), and PNO(n=8). In the piglets of the PCON and PNO groups, E. coli septic lung was induced by endotracheal instillation of E. coli. Ten ppm iNO was given continuously in the PNO group after endotracheal instillation of E. coli. All animals were mechanically ventilated for six hour with a peak inspiratory pressure of 30 cmH2O, frequency of 25 breaths/min, FiO2 1.0 and a positive end-expiratory pressure of 4 cmH2O. All measurements were made at one hour intervals during the experiment. At the end of the experiment, lung tissue was harvested for the analysis of myeloperoxidase activity, indicative of lung inflammation. RESULTS: All piglets with pulmonary instillation of E. coli developed E. coli sepsis. Piglets in the PCON group developed progresseve pulmonry hypertension, hypoxemia and hypercarbia compared to the CON group due to increased pulmonary vascular resistance, intrapulmonary shunt fraction and physiologic dead space fraction. iNO did not reverse pulmonary hypertension in the PNO group. However iNO significantly improved oxygenation, which was attributed to marked improvement of venous admixture and partial attenuation of increase in dead space fraction. Increased myeloperoxidase activity in PCON compared to CON was significantly attenuated in PNO. CONCLUSION: iNO improves oxygenation and lung inflammation in newborn piglets with E. coli induced septic lung.