The effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and formation of neutrophil extracellular traps in sepsis rats
10.3760/cma.j.issn.1671-0282.2022.03.014
- VernacularTitle:大鼠肺组织胞外囊泡对脓毒症肺内炎症及中性粒细胞胞外诱捕网形成的影响
- Author:
Fen LIU
1
;
Wei PENG
;
Yong WANG
;
Yuanlei LOU
;
Ning ZHAO
;
Kejian QIAN
;
Yong LI
Author Information
1. 南昌大学第一附属医院重症医学科，南昌 330006
- Keywords:
Tissue derived extracellular vesicles;
Isolation and extraction;
Septic lung injury;
Inflammation;
Neutrophil extracellular traps
- From:
Chinese Journal of Emergency Medicine
2022;31(3):338-343
- CountryChina
- Language:Chinese
-
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.
