Objective To investigate the effect of mesenchymal stem cell-derived extracellular vesicles(MSC-EV)on neutro-phil extracellular trap(NET)in sepsis-induced acute lung injury(SI-ALI)and the underlying mechanisms.Methods First,the ex-tracted MSC-EV were characterized:their size range was detected using nano-flow cytometry,morphology was observed by electron mi-croscopy,and EV markers[Apoptosis linked gene 2 interacting protein X(Alix),CD63,CD9,and Calnexin]were examined by Western blot.Eighteen wild-type C57BU6J mice were divided into three groups:a sham-operated(S)group,a cecal ligation and puncture(CLP)group,and a treatment(CME)group,with six mice in each group,according to random number table method.Mice in the S group under-went laparotomy without further treatment,followed by wound closure;those in the CLP group received CLP treatment;and the CME group was injected via the tail vein with MSC-EV(200 μg/mouse)1 h before CLP.Their lung pathologica changes was compared among the three groups by hematoxylin-eosin(H-E)staining.The levels of myeloperoxidase(MPO)and citrullinated histone H3(CitH3)in lung tissue were detected by immunofluorescence staining.Neutrophils in human peripheral blood samples were divided into three groups:a control(C)group,a phorbol myristate acetate(PMA)group,and a cell therapy(PME)group,according to random number ta-ble method.The C group was treated with phosphate-buffered saline(PBS)as a control;the PMA group was treated with PMA 50 nmol/L;and the PME group was treated with MSC-EV 20 mg/L 1 h before PMA stimulation.The levels of CitH3 in the three cell groups were compared by immunofluorescence staining,and reactive oxygen species(ROS)levels were compared using ROS staining.Results Nano-flow cytometry showed the MSC-EV particle size range was 60-100 nm,transmission electron microscopy showed cup-shaped vesicles,and Western blot showed that MSC-EV expressed EV markers Alix,CD63,CD9,but not Calnexin.The CLP group showed thicker alveolar walls,more hemorrhage,and more inflammatory cell infiltration in lung tissue,as well as higher MPO and CitH3 levels than the S group(all P＜0.05).Compared with the CLP group,the CME group showed decreases in alveolar walls,hemorrhage,and in-flammatory cell infiltration in lung tissue,as well as MPO and CitH3 levels(all P＜0.05).Compared with the C group,the PMA group showed increased CitH3 and ROS levels(all P＜0.05).Compared with the PMA group,the PME group presented reduced CitH3 and ROS levels(all P＜0.05).Conclusions MSC-EV can inhibit NET formation by reducing ROS,thereby alleviating SI-ALI.
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