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.