Animal experiments and ethical statement
All animal care and experimental procedures were approved by the Guangxi University of Chinese Medicine Animal Policy and Welfare Committee (Approval Document No. SYXK-GUI-2019-0001). All animals received humane care according to the Local Guide for the Care and Use of Laboratory Animals of Guangxi University of Chinese Medicine. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; Mcgrath & Lilley, 2015).
Male BABL/C mice weighing 18–22 g, were obtained from Hunan SJA Laboratory Animal Co. Ltd (Hunan, China). The mice were maintained on a 12 h light/dark cycle under specific pathogen-free conditions. Mice were acclimatized to the laboratory for 3 days, then randomly assigned to one of five groups (n=8 per group). The groups consisted of vehicle control (CON), an LPS-induced ALI group (4 mg·kg-1 or 15 mg·kg-1; intratracheally, i.t.), a dexamethasone treatment group (5 mg·kg-1; dexamethasone intraperitoneally, i.p.), a YDZG treatment of LPS-induced ALI (5 mg·kg-1; YDZG; intravenously, i.v.), and a second YDZG treatment of LPS-induced ALI (10 mg·kg-1; YDZG; intravenously, i.v.). LPS induction of ALI was performed according to a method previously described, with slight modifications (Spoelstra et al., 2007). Briefly, mice were anesthetized using pentobarbital sodium (48 mg·kg-1; i.p.), then mice treated with non-invasive intratracheal instillation of LPS into lung tissue (4mg·kg-1 or 15mg·kg-1; i.t.). Control mice received sterile saline. In the Dexamethasone group, mice were given dexamethasone (5 mg·kg-1; i.p.) 6 h after ALI induction. ALI + YDZG 5 group mice were given YDZG (5 mg·kg-1; i.v.) 2 h before ALI induction; Same concentration of YDZG was also given at 6 h and 12 h after ALI induction. ALI + YDZG 10 group mice were given YDZG (10 mg·kg-1; i.v.) 2 h before ALI induction, and were given YDZG 6 h and 12 h after ALI induction. Mice were sacrificed by cervical dislocation under ether anesthesia. Serum, bronchoalveolar lavage fluid (BALF, see below), and lung tissue samples were collected and stored at -80 °C. Serum and BALF samples were used for cytokine measurements via TNF-α, IL-6 and IL-1β ELISA kits. Lung homogenate was prepared from lung tissue and was used for cytokine detection by ELISA. Portions of lung sections were fixed in 4% paraformaldehyde and embedded in paraffin for histological analysis (see below). The remaining lung tissue was used for protein lysate preparation for immunoblotting.