Specific pathogen-free male C57BL/6N mice (20~25 g, six to eight weeks-old) were purchased from the Samtako Co. (Osan, Korea). They were housed in groups of nine under standard conditions (temperature 22±2℃, humidity 55±5%, 12-h-light/dark cycle) with food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chonnam National University.

The CS was generated from 3R4F research cigarette (Kentuchy reference cigarette, University of Kentuchy, USA), containing 11.0 mg of total particulate matter, 9.4 mg of tar, and 0.76 mg of nicotine per cigarette. Exposure to CS (one puff/min, 35 mL puff volum over 2 seconds, every 60 seconds, 8 cigarettes per day) was conducted using cigarette smoke generator (Daehan Biolink, Republic of Korea). The mice were exposed to CS for 1 h in a chamber (50 cm×30 cm×30 cm) for 28 days. LPS were intranasally instilled (10 µg dissolved in 50 µL distilled water) under anesthesia on day 26. The HemoHIM was obtained from Korea Institute of Oriental Medicine (Daejeon, Republic of Korea). HemoHIM was administered to mice at doses of 50 or 100 mg/kg by oral gavage 1 h before CS exposure for 28 days. A positive control group was administered roflumilast (Sigma-Aldrich, St, Louis, MO, USA, 10 mg/kg) which is a PDE-4 inhibitor and manufactured for treatment COPD.

Forty-eight hours after the last intranasal LPS administration, the mice were sacrificed via an intraperitoneal injection of zoletil 50 (25 mg/kg; Virbac korea. Co., Seoul, Korea), and a tracheostomy was performed according to a previous study [19]. To obtain the broncho-alveolar lavage fluid (BALF), ice-cold PBS (0.7 mL) was infused into the lung and withdrawn via tracheal cannulation. This process was repeated once (total volume 1.4 mL). To determine the differential cell counts, 100 µL of BALF was centrifuged onto slides using a Cytospin (Hanil Science Industrial, Seoul, Korea). The slides were dried, and the cells were fixed and stained using Diff-Quik staining reagent (B4132-1A; IMEB Inc., Deerfield, IL) according to the manufacturer's instructions. The supernatant obtained from the BALF was stored at −70℃ for biochemical analysis.

The pro-inflammatory mediators in the BALF were measured using ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer's protocols. The plates were incubated for 10 min in the dark, and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, Laboratories).

The lung tissue was homogenized (1/10 w/v) using a homogenizer in a Tissue Lysis/Extraction reagent (Sigma-Aldrich, St, Louis, MO, USA) that contained a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using Bradford reagent (Bio-Rad). Equal amounts of the total protein (30 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with blocking solution (5% skim milk) followed by overnight incubation at 4℃ with the appropriate primary antibody. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, Danvers, MA, USA), anti-pERK (1:1000 dilution; Cell Signaling), anti-ERK (1:1000 dilution; Cell Signaling) and anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, MA, USA). The blots were washed three times with Tris-buffered saline containing Tween 20 (TBST) and then incubated with a 1:10000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA) for 30 min at room temperature. The blots were then washed three times with TBST and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Carlsbad, CA, USA).

SDSPAGE zymography was performed according to previous study (Shin et al., 2014) to determine gelatinase activitiy. Briefly, zymogram gels comprised of 10% SDS-PAGE containing 1% gelatin were used as the MMP substrate. The gels were washed in 2.5% Triton X-100 for 1 h to remove SDS and then incubated at 37℃ for 16 h in developing buffer (1M Tris-HCl, pH 7.5 with CaCl₂). Thereafter, gels were stained with 25% methanol/8% acetic acid containing Coomassie Brilliant Blue. Gelatinase activity was visualized as white bands on a blue background that represented the areas of proteolysis.

The lung tissue was fixed in 4% (v/v) paraformaldehyde, embedded in paraffin, sectioned at 4-µm thickness, and stained with hematoxylin and eosin (H&E_solution; Sigma-Aldrich) to estimate inflammation.

Immunohistochemical slides were deparaffinized, dehydrated, washed in PBS containing 0.05% tween 20 (PBS-T), and incubated for 20 min at room temperature with goat serum to block nonspecific staining. The slides were incubated for 2 h at room temperature with primary mouse anti-mouse MMP-9 antibody (diluted 1:100, Abcam). After incubation, they were washed three times, incubated for 1 h at room temperature with a biotinylated secondary antibody, and then incubated with an avidinbiotin-peroxidase complex (Vector Laboratories, Burlin-game, CA, USA) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5min.

The data are expressed as the means±standard deviation (SD). Statistical significance was determined using an analysis of variance (ANOVA) followed by a multiple comparison test with Dunnet's adjustment. P values <0.05 were considered significant.

The number of inflammatory cells in BALF was increased in CS and LPS exposed mice compared with vehicle control mice. Specifically, CS and LPS exposure markedly increased the number of neutrophils in BALF compared to control. In HemoHim treated mice, however, the number of neutrophils in BALF decreased in a dose-dependent manner compared to CS and LPS exposed mice (Figure 1).

In CS and LPS exposed mice, the levels of TNF-α in BALF were significantly increased compared to the vehicle control mice (Figure 2A). Roflumilast significantly reduced TNF-α in CS and LPS exposed mice. In addition, HemoHim treated mice showed a dose-dependent decrease TNF-α compared to CS and LPS exposed mice. The results of IL-6 and IL-1β in BALF were similar to those of TNF-α (Figure 2A, B). CS and LPS exposure mice significantly increased level of IL-6 and IL-1β in BALF compared with the vehicle control mice, and HemoHim treated mice significantly decreased the level of IL-6 and IL-1β in BALF in CS and LPS exposed mice.

iNOS expression increased in the lung tissue of CS and LPS exposed mice compared to vehicle control mice. Roflumilast treated mice markedly decreased iNOS expression in the lung tissue compared with CS and LPS exposed mice. In addition, HemoHIM treated mice showed a dose-dependent decrease of iNOS expression in lung tissue compared to CS and LPS exposed mice (Figure 3A).

In comparison to vehicle control mice, the phosphorylation of Erk was significantly increased in CS and LPS exposed mice. HemoHIM markedly and dose-dependently decreased phosphorylation of Erk in CS and LPS exposed mice (Figure 3B).

CS and LPS exposed mice exhibited extensive inflammatory cell infiltration into the lung tissue (Figure 4). Inflammatory cells mainly accumulating in peribronchial and alveolar lesions. In contrast, roflumilast treated mice decreased inflammatory cell infiltration into lung tissue induced by CS and LPS exposure. Similarly, inflammatory cell infiltration was significantly reduced in a dose-dependent manner in HemoHim treated mice compared to CS and LPS exposed mice.

MMP-9 expression in lung tissue was markedly increased in CS and LPS exposed mice compared to vehicle control mice (Figure 5A). HemoHim treated mice, however, reduced this increased expression of MMP-9 in lung tissue induced by CS and LPS exposure. In zymographs, CS and LPS exposed mice showed a marked increase in MMP-9 activity compared with the vehicle control mice, whereas HemoHim treated mice exhibited a marked and dose-dependent reduction in MMP-9 activity compared with CS and LPS exposed mice (Figure 5B).