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

The effect of extracellular vesicles derived from lung tissue on intrapulmonary infl ammation and formation of neutrophil extracellular traps in sepsis rats

目的探讨脓毒症大鼠肺组织来源的胞外囊泡对脓毒症肺内炎症反应及中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)形成的影响。方法采用盲肠结扎穿孔术(cecal ligation and puncture,CLP)建立脓毒症大鼠模型,利用Collagenase D和DNase I充分酶解消化肺组织,多次离心去除杂质,最后通过差速超速离心法提取肺组织胞外囊泡(Ti-EVs)。将18只雄性SD大鼠随机分为3组:Ti-EVs组行CLP术并经气道滴入Ti-EVs混悬液(2.5 mg/kg);CLP组行CLP术并经气道滴入等体积PBS;Sham组行假手术。24 h后利用苏木精-伊红(HE)染色检测肺组织病理学变化,酶联免疫吸附实验检测肺泡灌洗液(bronchoalveolar lavage fl uid,BALF)中IL-1β、TNF-α及IL-6的表达水平,免疫荧光法检测肺组织内NETs含量。结果分离提取的大鼠肺组织胞外囊泡经透射电镜观察为双层圆形的囊性小泡结构,粒子直径主要分布在150 nm,Western blot显示EVs标志物CD9、CD63、Tsg101呈阳性表达。肺组织HE染色可见脓毒症大鼠肺泡完整性破坏,炎症细胞浸润。与CLP组相比,Ti-EVs组肺组织损伤程度更重,且BALF中IL-1β、TNF-α、IL-6炎症因子表达水平均显著升高(P<0.01);激光共聚焦显微镜下见脓毒症组和Ti-EVs组肺内均有NETs形成,Ti-EVs组肺内NETs荧光强度较脓毒症组明显增加。结论通过酶解消化、差速超速离心等系列步骤能够成功分离提取得到纯度较高的大鼠肺组织胞外囊泡。在脓毒症肺损伤进程中,肺组织胞外囊泡可加重肺内炎症反应,促进NETs形成。

Objective To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary infl ammation 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 fi nally,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 interleukin1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the bronchoalveolar lavage fl uid(BALF)was measured by enzyme-linked immunosorbent assay(ELISA).Immunofl uorescence 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 infl ammatory cells infi ltrated 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-αand IL-6 in the BALF of rats were signifi cantly 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 fl uorescence intensity of NETs in the lung tissues of the Ti-EVs group increased signifi cantly.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 infl ammatory response in the lung and promote the formation of NETs.