The ARDS model was established as described previously (40 (link)). Briefly, the mice were anesthetized with sevoflurane (Hengrui, Lianyungang, Jiangsu, China). After exposing the trachea, a trimmed sterile 31-gauge needle was inserted into the tracheal lumen. LPS (Sigma, St Louis, MO, USA) diluted in endotoxin-free saline was intratracheally (IT) injected at a dose of 10 mg/kg in 100 μl saline. To treat ARDS mice, anti-PD-L1 antibody (eBiosciences, San Diego, CA, USA) was intraperitoneally administered at a dose of 50 μg/mouse immediately after the injection of LPS. Hematoxylin-eosin staining was conducted to quantify lung injury and the result was semi-quantified by two independent pathologists according to the criteria reported previously (41 (link), 42 (link)). The cells in the BALF (BALF was obtained by intratracheal injection with 1 ml cold PBS) were collected and stained with anti-Ly6G-PE and anti-CD11b-APC (eBiosciences, San Diego, CA, USA) to detect neutrophils by flow cytometry. BALF levels of TNF-α, IL-1β and IL-6 were detected by enzyme linked immunosorbent assay (ELISA, R&D, Minneapolis, MN, USA). The wet-to-dry weight (W/D) ratio was calculated to assess the edema. The protein concentration in the BALF was assessed with a BCA detection kit (Thermo Scientific, Rockford, IL, USA).
Free full text: Click here
