LPS-induced lung inflammation in mice

For measuring in vivo therapeutic effects, LPS-induced acute lung injury (ALI), a mouse model of airway inflammation, was used (Lim et al., 2013). The test compounds, including the reference drug, were dissolved in 0.3% carboxymethylcellulose (CMC) and were orally administered to mice at the indicated doses (n=10). The control and LPS treatment groups also received the same amount of CMC solution. For inducing bronchitis, LPS (E. coli 0127:B8, 2 mg/kg, PBS) was administered intranasally to mice (10 μl/mouse, 5 times) at 1 h after oral treatment with the test compounds according to the previously published procedures (Lim et al., 2013). At 16 h after LPS treatment, mice were sacrificed (n=7), and bronchoalveolar lavage fluid (BALF) was collected via intratracheal cannulation. In the BALF, the total cell number was counted with a haemocytometer, and the cells were differentially counted with fluorescence-activated cell sorter (FACS, BD Biosciences, San Jose, CA, USA). For FACS analysis, cells were stained with following antibodies; allophycocyanin (APC) conjugated anti-Ly-6C (BD Bioscience), allophycocyanin-Cy7 (APC-Cy7) conjugated anti-CD11c (BD Bioscience), fluorescein isothiocyanate (FITC) conjugated anti-CD11b (BD Bioscience), phyco-erythrin (PE) conjugated anti-F4/80 (eBioscience, San Diego, CA, USA), phycoerythrin–Cy7 (PE-Cy7) conjugated anti-Ly-6G (BD Bioscience). After incubation with antibody, cells were washed with 2% FBS RPMI media (Thermo Fisher Scientific, Waltham, MA, USA). Cell pellets were resuspended and analyzed. CD11b, Ly-6C and Ly-6G are essential markers of neutrophils. CD11c and F4/80 are markers of macrophages. For the histological analysis, the remaining mice (n=3) were sacrificed and lung tissues were excised. Histological examination was carried out using the conventional methods of H&E staining. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure the thickness of alveolar wall area.