Canarium lyi C.D. Dai & Yakovlev of the Burseraceae family was collected from the area of Gia Lai, K Bang, So Pai, Vietnam in 2011. Plant samples were identified by Dr Tran The Bach of the Institute of Ecology and Biological Resources. A voucher specimen (KRIB 0036679) was deposited in the herbarium (KRIB) of the Korea Research Institute of Bioscience and Biotechnology. Canarium lyi (147 g) was treated with MeOH and sonicated several times at room temperature for 3 days to produce an extract (20.05 g).

RAW 264.7 cells were maintained at 1×10⁵ cells/ml in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Burlington, ON, Canada), and 1% (w/v) of an antibiotic-antimycotic solution (Invitrogen, Grand Island, NY, USA) in 95% air and 5% CO₂ humidified atmosphere at 37°C.

Cell viability was determined by assessing the mitochondrion-dependent reduction of MTT (Amresco LLC, Solon, OH, USA) to formazan. Briefly, 5 μl of a 5 mg/ml MTT solution was added to the cell supernatant, and then incubation for 4 h at 37°C. DMSO was added following removal of the medium. The optical density of formazan was measured using a microplate reader (VersaMax; Molecular Devices, San Diego, CA, USA) at 570 nm. The level of formazan generated by untreated cells was chosen as the 100% value.

Nitrite levels in the cultured media and serum, which reflect intracellular nitric oxide (NO) synthase activity, were determined by Griess reaction. The cells were incubated with samples in the presence of LPS (0.5 μg/ml, Sigma-Aldrich, La Jolla, CA, USA) at 37°C for 24 h. The cell supernatant was dispensed into new 96-well plates, and 100 μl of each supernatant was mixed with the same volume of the Griess reagent [1% sulfanilamide, 0.1% N-(1-naphathyl)-ethylenediamine dihydrochloride and 5% phosphoric acid] and incubated at room temperature for 10 min. Sodium nitrite was used to generate a standard curve, and the concentration of nitrite was measured for absorbance at 540 nm.

The levels of IL-6 in supernatant were determined using a commercially available ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. IL-6 levels were determined from a standard curve. The concentrations were expressed as pg/ml.

PGE₂ levels in supernatants were determined using a PGE₂ EIA kit (Cayman Chemical Co., Inc., Ann Arbor, MI, USA) according to the manufacturer’s instructions. Briefly, 50 μl diluted standards/samples were pipetted into the wells of a 96-well plate precoated with goat polyclonal anti-mouse IgG. Aliquots of a PGE₂ monoclonal antibody solution and a PGE₂ acetylcholine esterase conjugate solution were added to each well, and incubated at room temperature for 18 h. The wells were washed six times with a wash buffer containing 0.05% (v/v) Tween-20, followed by the addition of 200 μl Ellman’s reagent containing acetylthiocholine and 5,5′-dithio-bis-(2-nitrobenzoic acid). PGE₂ concentrations were measured by absorbance at 405 nm.

Total RNA was isolated using TRIzol™ reagent (Life Technologies Corp., Carlsbad, CA, USA). For RT-PCR, a single-strand cDNA was synthesized from 2 μg total RNA. The primer sequences used were: iNOS: sense, 5′-CAA GAG TTT GAC CAG AGG ACC-3′ and antisense, 5′-TGG AAC CAC TCG TAC TTG GGA-3′; COX-2: sense, 5′-GAA GTC TTT GGT CTG GTC TCC TG-3′ and antisense, 5′-GTC TGC TGG TTT GGA ATA GTT GC-3′; TNF-α: sense and 5′-CAT CTT GCA AAA TTC GAG TGA CAA-3′ and antisense, 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; IL-6, sense, 5′-GAG GAT ACC ACT CCC AAC AGA CC-3′ and antisense, 5′-AAG TGC ATC ATC GTT GTT CAT ACA-3′, β-actin: sense, 5′-CGC TCA TTG CCG ATA GTG AT-3′ and antisense 5′-TGT TTG AGA CCT TCA ACA CC-3′. PCR products were fractionated on 1.5% agarose gel electrophoresis and stained with 5 μg/ml ethidium bromide. Images were captured by an Olympus C-4000 Zoom camera system (Olympus America Inc., Center Valley, PA, USA).

Western blot analyses were performed as previously described (14). Immunoblotting was performed with the primary antibodies at 4°C overnight. A horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was then used for 1 h. The membranes were washed three times with TBST, and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, San Jose, CA, USA). For quantitative analysis, densitometric band values were determined using a bio-imaging analyzer (LAS 4000 mini; Fujifilm, Tokyo, Japan).

Male C57BL/6 mice (6–8 weeks) were obtained from the Koatech Co. (Pyeongtaek, Korea). The mice were fed with food and water ad libitum in an animal facility with temperature ranging from 22 to 24°C and a 12-h light/dark cycle under a specific pathogen-free conditions. Prior to the initiation of the experiment, the mice were housed for a minimum of one week in order that they adapt to the environment. The animal experimental procedures were approved by the Korea Research Institute of Bioscience and Biotechnology and performed in compliance with the National Institute of Health Guidelines for the care and use of laboratory animals and the Korean national animal welfare law.

Mice were randomly allocated into 4 groups: Control, LPS, LPS + dexamethasone (LPS + DEX) and LPS + CL. Dexamethasone served as a positive control drug. The mice of the LPS + DEX and LPS + CL groups received dexamethasone (3 mg/kg) and CL (30 mg/kg) by oral gavage for 3 days, respectively. The mice from the control and LPS groups received oral gavage at an equal volume of PBS. The mice including the LPS, LPS + DEX and LPS + CL groups were instilled with 10 μg of LPS dissolved in 50 μl PBS intranasally to induce acute lung injury 1 h after final drug treatment. Mice in the control group were intranasally given 50 μl PBS without LPS for 18 h. To obtain BALF, ice-cold PBS (0.7 ml) was infused into the lung and withdrawn via tracheal cannulation twice (total volume 1.4 ml).

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 the differential cell counts, 100 μl of BALF was centrifuged onto slides using a Cytospin (Hanil Science Industrial Co., Ltd., Seoul, Korea) (200 × g for 5 min). The slides were dried, and the cells were fixed and stained using a Diff-Quik^® staining reagent (B4132-1A; IMEB Inc., Deerfield, IL, USA) following the manufacturer’s instructions. The supernatant obtained from BALF was stored at −70°C for the biochemical analysis.

Lung tissue was homogenized (1/10 w/v) using a homogenizer with a tissue lysis/extraction reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Each protein concentration was determined using a Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting was performed as described above, and the levels of COX-2, IκB-α and NF-κB were determined.

The levels of IL-6 (R&D Systems), TNF-α and IL-1β (BD Biosciences, San Jose, CA, USA) in BALF were measured using ELISA kits according to the manufacturer’s instructions.

After BALF samples were obtained, lung tissues were fixed in 4% (v/v) paraformaldehyde. Tissues were embedded in paraffin, sectioned at 4 μm thickness, and stained with H&E solution (Sigma-Aldrich) to estimate inflammation.

Data are expressed as the means ± the standard error of the mean (SEM). Statistical significance was determined using analyses of variance (ANOVAs) followed by multiple comparison tests with Dunnet’s adjustment. P<0.05 was considered significant.

We determined the effect of CL on cell viability by MTT assay after incubating cells for 24 h. The cytotoxicity of CL was preliminarily evaluated to establish the appropriate concentration ranges of CL for the analysis of ongoing experiments. Results showed that the cells’ viabilities were not affected by CL at the concentrations (5, 10, 20 and 40 μg/ml) used (Fig. 1A). Therefore, we used non-toxic concentrations (5–40 μg/ml) for the entire experiment.

LPS-stimulated cells markedly increased in NO, whereas CL treatment significantly decreased NO production in a concentration-dependent manner (Fig. 1B). We also examined the effects of CL on PGE₂ production following LPS stimulation in RAW 264.7 cells. The amount of PGE₂ was increased by the LPS stimulation in the culture supernatant, and this increase was effectively reduced by treatment with CL (Fig. 1C).

Similarly, treatment of the RAW 264.7 cells with LPS alone resulted in a significant increase in cytokine production compared with the control group (Fig. 1D). However, CL treatment considerably inhibited the LPS induction of IL-6 in a dose-dependent manner (Fig. 1D).

The production of mRNA and protein of iNOS, COX-2 and pro-inflammatory cytokines, including IL-6 and TNF-α, increased in LPS-stimulated RAW 264.7 cells (Fig. 2). However, treatment of the cells with CL significantly decreased iNOS, COX-2 and pro-inflammatory cytokine production compared to the LPS-stimulated cells in a concentration-dependent manner (Fig. 2).

LPS-stimulated cells showed an increase in the phosphorylation levels of p38, ERK1/2 and JNK. By contrast, treatment of the cells with CL significantly reduced the phosphorylation of p38, ERK and JNK expression compared with the LPS-stimulated cells in a concentration-dependent manner (Fig. 3).

We examined the effect of CL on LPS-stimulated IκB-α degradation. One hour of pre-treatment with CL followed by treatment with LPS for 30 min markedly suppressed the LPS-stimulated phosphorylation and degradation of IκB-α in a dose-dependent manner (Fig. 4A). We also investigated the translocation of the NF-κB subunit p65 from the cytosol to the nucleus using western blot analysis. LPS stimulation caused p65 translocation from the cytosol to the nucleus, while CL inhibited this translocation (Fig. 4B).

The LPS group showed a significant increase in the number of total inflammatory cells and neutrophils in BALF compared with the negative control group. However, pre-treatment with CL significantly decreased the number of total inflammatory cells and neutrophils compared with the LPS group (Fig. 5).

The levels of IL-6, IL-1β and TNF-α in BALF were significantly higher in LPS-induced mice when compared with the negative control group. By contrast, the CL-treated mice had considerably lower levels of all these pro-inflammatory cytokines compared to the LPS-treated mice (Fig. 6).

The LPS-induced mice exhibited an increased expression of COX-2, p-IκB and p-p65 in the lung tissue compared to the negative control group. However, treatment of the mice with CL significantly inhibited the expression of COX-2, IκB degradation and the phosphorylation of p65 in the lung tissue compared to the LPS-treated mice (Fig. 7).

We confirmed an intact structure and clear pulmonary alveoli in the lung sections of mice in the negative control group. By contrast, lung sections obtained from mice in the LPS group showed evidence of histological changes, including areas of inflammatory cell infiltration, thickening of the alveolar wall, edema and pulmonary congestion. By contrast, treatment of the mice with CL attenuated the pathological changes that were observed in the LPS-treated mice (Fig. 8).