RAW 264.7 macrophages were maintained at 1×10⁵ cells/ml in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Sigma-Aldrich, St. Louis, MO, USA) and 1% (w/v) antibiotic-antimycotic solution (Invitrogen, Grand Island, NY, USA) in a 95% air and 5% CO₂ humidified atmosphere at 37°C. The cells were seeded in 96-well plates at 5×10⁴ cells/well and incubated in serum-free medium in the presence of different CA concentrations (5, 10, 20 and 40 µg/ml). CA was obtained from the Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) (BVN12106; Daejeon, Korea).

Cell viability was evaluated by determining the mitochondrial reductase function using with an assay based on the reduction of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (CAS#298-93-1; Amresco, LLC, Solon, OH, USA) into formazan crystals. Formazan formation is proportional to the number of functional mitochondria in living cells. To determine cell viability, 50 mg/ml of MTT was added to 5 µl of cell suspension (1×10⁵ cells/ml in 96-well plates) for 4 h. The formazan formed was dissolved in acidic 2-propanol, and the optical density was measured at 570 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The optical density of formazan formed in the control (untreated) cells was set to 100% viability.

NO production in RAW 264.7 cell culture supernatants was determined using the Griess reaction. The cells were incubated in the presence of LPS (0.5 µg/ml) at 37°C for 24 h. The cells were then dispensed into 96-well plates, and 100 µl of each supernatant was mixed with the same volume of Griess reagent (1% sulfanil-amide, 0.1% N-(1-naphathyl)-ethylenediamine dihydrochloride and 5% phosphoric acid) and agitated gently for 10 min at room temperature. The nitrite concentration was measured at 540 nm using sodium nitrite to generate a standard curve.

Cultures were serum-starved for 24 h and treated with experimental agents. Following culture incubation under experimental conditions, the media from each sample were collected and assessed for IL-6 synthesis using a commercial enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol, and the assay was calibrated spectrophotometrically with a standard curve. Supernatant PGE₂ levels were determined using a PGE₂ enzyme immuno assay (EIA) kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocol.

Total RNA was reverse transcribed using AccuPower RT Premix (Bioneer Corporation, Daejeon, Korea) according to the manufacturer's instructions. The resulting cDNA (equivalent to 40 ng total RNA) was used for qPCR using FastStart DNA Master^PLUS SYBR-Green I and LightCycler (both from Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. qPCR was performed using the primers: COX-2, sense, 5′-GAAGTCTTTGGTCTGGTCTCCTG-3′ and antisense, 5′-GTCTGCTGGTTTGGAATAGTTGC-3′; and β-actin sense, 5′-CGCTCATTGCCGATAGTGAT-3′ and antisense 5′-TGTTTGAGACCTTCAACACC-3′. All of the samples were normalized to β-actin. qPCR products were subjected to electrophoresis on 1.5% agarose gels and stained with ethidium bromide. Images were captured with an Olympus C4000 zoom camera system (Olympus America Inc., Melville, NY, USA).

Cells (1×10⁶) were harvested and washed twice in cold Tris-buffered saline (TBS). The cells were solubilized in ice-cold 1% Triton X-100 lysis buffer. After 30 min on ice, the lysates were clarified by centrifugation at 600 × g for 20 min. Proteins (20 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide) and transferred to nitrocellulose membranes. The membranes were incubated with blocking solution (5% skim milk) and then incubated overnight at 4°C with the appropriate primary antibodies. The primary antibodies and dilutions used were: anti-β-actin (1:2,000 dilution; Cat. no. P60709; Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-COX-2 (1:1,000 dilution; Cat. no. SC-1745; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were washed three times with TBS containing Tween-20 (TBST) and then incubated with a 1:3,000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies (Cat. no. SC-2027; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The membranes were again washed three times with TBST and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Waltham, MA, USA). For quantitative analysis, densitometric band values were determined using ChemiDoc (Bio-Rad Laboratories, Inc.).

Male C57BL/6 mice (6–8 weeks of age) were obtained from Orient Co. (Seoul, Korea). The mice were provided with food and water ad libitum in an animal facility at a temperature of 22–24 °C and on a 12 h light/dark cycle under specific pathogen-free conditions. The mice were housed for a minimum of one week for environmental adaptation prior to experimentation. Experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology.

Specific pathogen-free male C57BL/6 mice were randomly divided into four groups (n=7/group): i) NC (normal control); ii) LPS (LPS treatment); iii) Dex (LPS treatment + oral gavage of 5 mg/kg dexamethasone for 3 days); and iv) CA (LPS treatment + oral gavage of 30 mg/kg CA for 3 days). The mice from the control and LPS groups received an equal volume of phosphate-buffered saline (PBS) instead of dexamethasone or CA. Three hours after drug administration on the third day, the mice were slightly anesthetized with diethyl ether inhalation, and 10 µg of LPS in 50 µl of PBS was instilled intranasally (i.n.) to induce lung injury. The mice in the control group were given 50 µl of PBS without LPS. Twenty-four hours after the LPS treatment, the mice were sacrificed by an i.p. injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co. Ltd., Seoul, Korea) and bronchoalveolar lavage fluid (BALF) and lung tissue samples were harvested for further studies.

BALF collection was performed three times through a tracheal cannula with auto-claved PBS and instilled up to a total volume of 1.4 ml. The BALF was centrifuged at 200 × g for 15 min at 4°C. Following centrifugation, the supernatant was stored at −70°C for subsequent cytokine assays. The total inflammatory cell numbers were assessed by counting cells in at least five squares of a hemocytometer after excluding dead cells by trypan blue staining. To determine differential cell counts, 100 µl of BALF was centrifuged onto slides using a Cytospin (Hanil Science Industrial, Seoul, Korea). After the slides were dried, the cells were fixed and stained using Diff-Quik^® staining reagent (B4132-1A; IMEB Inc., Deerfield, IL, USA).

Detection of cellular ROS in inflammatory cells was performed using 2′,7′-dichlorofluorescein diacetate (DCF-DA) reagent as part of a kit purchased from Abcam (ab112851; Cambridge, MA, USA). After cell counting, 100 µl of inflammatory cells were seeded in 96-well plates. The cells were stained with 20 µM DCFDA for 30 min at 37°C. Fluorescence intensity was then measured at 485 nm excitation and 530 nm emission using a microplate reader (Bio-Rad Laboratories, Inc.).

TNF-α, IL-1β and IL-6 levels in BALF were measured using ELISA kits (BioSource International, Camarillo, CA, USA). The procedures were performed according to the manufacturer's instructions.

After obtaining the BALF samples, lung tissue was fixed in 10% (v/v) neutral-buffered formalin. Lung tissues were embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin (H&E) solution (both from Sigma-Aldrich; hematoxylin, MHS-16; eosin, HT110-1-32) to estimate inflammation.

Lung tissues were harvested and homogenized (1/10 w/v) using a homogenizer and tissue lysis/extraction reagent containing a protease inhibitor cocktail (both from Sigma-Aldrich). Protein concentrations were determined using a Bradford reagent (Bio-Rad Laboratories, Inc.,). Western blotting was performed as described above, and the following primary antibodies and dilutions used were: anti-IκB (1:1,000 dilution; Cell Signaling Technology, Inc.,), anti-NF-κB/p65 (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) and anti-β-actin (1:2,000 dilution; Cell Signaling Technology, Inc.).

The photomicrographs were obtained using a Photometric Quantix digital camera running Windows, and images were assembled in Adobe Photoshop 7.0. Images were cropped and corrected for brightness and contrast but were not otherwise manipulated.

Data were presented as the means ± standard error of the mean (SEM). Statistical significance was determined using analysis of variance (ANOVA) followed by a multiple comparison test with Dunnett's adjustment. P<0.05 was considered to indicate a statistically significant difference.

Non-toxic CA concentrations (5, 10, 20 and 40 µg/ml) (Fig. 1A) were assessed for their ability to inhibit LPS-stimulated NO production. NO production was significantly increased in LPS-stimulated RAW 264.7 cells. By contrast, CA at 20 and 40 µg/ml induced a significant decrease in LPS-induced NO production; NO production decreased in a concentration-dependent manner (Fig. 1B).

PGE₂ levels in the culture supernatant increased with LPS stimulation, and this effect was markedly reduced by CA treatment (Fig. 2A). Treatment of RAW 264.7 cells with LPS alone resulted in a significant increase in IL-6 production compared with the control group (Fig. 2B). However, CA treatment significantly inhibited LPS induction of IL-6, in a dose-dependent manner.

COX-2 mRNA and protein production was significantly increased in LPS-stimulated RAW 264.7 cells (Fig. 3). However, CA-treated cells exhibited significantly decreased COX-2 mRNA and protein levels compared with LPS-stimulated cells in a concentration-dependent manner.

The total number of inflammatory cells in the BALF comprised neutrophils, macrophages and lymphocytes. LPS challenge significantly increased the number of total cells, neutrophils, macrophages and lymphocytes compared with the control group. However, BALF samples from the CA-treated mice exhibited a significantly decreased influx of total cells, neutrophils, macrophages and lymphocytes compared with the LPS-treated mice (Fig. 4). The reductions were similar to the results obtained in the dexa methasone-treated mice.

ROS levels in the BALF were significantly increased in LPS-treated mice compared with the negative controls group (Fig. 5). By contrast, the CA-treated mice exhibited a significant decrease in ROS production compared with the LPS-treated mice. The reductions were similar to the results obtained for the dexamethasone-treated mice.

TNF-α, IL-1β and IL-6 levels in the BALF were significantly increased in LPS-treated mice compared with the negative control group (Fig. 6). By contrast, CA-treated mice exhibited a significant decrease in TNF-α, IL-1β and IL-6 levels compared with the LPS-treated mice. The reductions were similar to the results obtained in the dexamethasone-treated mice.

To evaluate histological changes following LPS treatment of LPS-induced lung inflammation, the lung sections were subjected to H&E staining. Significant pathological changes were observed in the lungs of LPS-treated mice including inflammatory cell infiltration, lung tissue damage, and alveolar wall thickening. However, fewer histopathological changes were observed in the lung tissues of CA-treated mice compared with the LPS-treated mice (Fig. 7). The reductions were similar to the results obtained in the dexamethasone-treated mice.

Mice with LPS-induced ALI exhibited increased IκB and NF-κB phosphorylation in the lung tissue samples compared with the negative control group. However, there was significantly decreased IκB and NF-κB phosphorylation in the lung tissue of CA-treated mice compared with the LPS-treated mice (Fig. 8). The reductions were similar to the results obtained in the dexamethasone-treated mice.