PQ was collected from Baegunsan Mountain of Gwangyang-si, Jeollanam-do, Korea. A voucher specimen recorded as KRIB 0001101 has been deposited at the herbarium of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). After drying and grinding the bark of the stem of PQ, the powder (50 g) was added to 100 liters of methanol. The extraction was performed using the method of repercolation at room temperature. The extract was filtered and concentrated using a rotary evaporator (N-1200AV-W; T.R.K. EYELA, Tokyo, Japan) under reduced pressure, thereby obtaining 2.07 g of PQ methanolic extract. In the following experiment, PQ was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/ml, and then diluted to various concentrations prior to use.

Specific pathogen-free male C57BL/6N mice (6 weeks old) were obtained from Koatech Co. (Pyeongtaek, Korea) and used after 2 weeks of quarantine and acclimatization. They were housed in groups of 7 under standard conditions with food and water. Briefly, the mice (n=7/group) were divided into 4 groups as follows: i) the normal control (NC) group; ii) the LPS group; iii) the dexamethasone (DEX; 3 mg/kg) group (used as a positive control); and iv) the PQ group (administered 5 and 15 mg/kg PQ). PQ and DEX were dissolved with 0.5% carboxymethyl cellulose (CMC), and were administered orally from day 0 to day 2. The mice were exposed to LPS (10 µg/mouse) intranasally 1 h after the final DEX and PQ treatment, as previously described (3). All the experimental procedures were performed in accordance with the procedures approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology and performed in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and Korean National Laws for Animal Welfare.

To obtain the BALF, ice-cold PBS (0.7 ml) was infused into the lungs of the mice twice and withdrawn each time using a tracheal cannula (a total volume of 1.4 ml). The collected BALF was centrifuged for 5 min at 1,500 rpm and the BALF differential cell count was determined using Diff-Quik^® staining reagent according to themanufacturer's instructions, and as previously described (3).

The intracellular levels of ROS were determined using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma Aldrich, St. Louis, MO, USA). Briefly, BALF cells were washed with PBS and incubated with 20 µM DCFH-DA for 10 min at 37°C. The activity of intracellular ROS was then detected by measuring the fluorescence at 488 nm excitation and 525 nm emission on a fluorescence plate reader (Perkin-Elmer, Waltham, MA, USA). The levels of pro-inflammatory cytokines (TNF-α and IL-6) in BALF were measured using ELISA kits according to themanufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Blood was collected from the inferior vena cava, and the levels of IL-6 in serum were determined using ELISA following themanufacturer's instructions (R&D Systems). The absorbance was measured at 450 nm was determined using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

The lung tissue was homoge-n-ized (1/10 w/v) using a homogenizer with tissue lysis/extraction reagent, containing a protease inhibitor cocktail (both from Sigma-Aldrich). Equal amounts of the total cellular protein (30 µg) were resolved by 12% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The membrane was blocked by incubation with 5% skim milk in TBST for 1 h, and incubated overnight at 4°C with the appropriate primary antibody. Specific antibodies against Nrf2 (ab137550, 1;1,000; Abcam, Cambridge, MA, USA), HO-1 (ab137749, 1;1,000; Abcam) p-p38 (1:1,000; ADI-KAP-MA022, 1:1,000; Enzo Life Sciences, Farmingdale, NY, USA), p38 (sc-7149, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK (#9106, 1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), ERK (sc-154, 1:1,000; Santa Cruz Biotechnology), p-JNK (KAP-SA011, 1:1,000; Enzo Life Sciences), JNK (sc-474, 1:1,000), IκB (sc-371, 1:1,000), p65 (sc-372, 1,000) (all from Santa Cruz Biotechnology), p-p65 (#3033, 1:1,000; Cell Signaling Technology Inc.), iNOS (ADI-905-431, 1:1,000; Enzo Life Sciences) and β-actin (#4967, 1:2,500; Cell Signaling Technology Inc.) were diluted in 5% skim milk. The blots were washed with TBST and incubated with a 1:2,000 dilution of a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. The blots were washed with TBST and developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The protein bands were visualized using a LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan) and quantified by densitometry (Fuji Multi Gauge software version 3.0).

RAW 264.7 murine macrophages (2×10⁵ cells/ml; ATCC, Manassas, VA, USA) were cultured in 6-well plates, treated with PQ for 1 h and stimulated with 0.5 µg/ml of LPS. The cells were harvested and washed twice with ice-cold PBS. Nuclear and cytoplasmic fractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA), according to themanufacturer's instructions. The nuclear translocation of NF-κB and Nrf2 was determined by western blot analysis. PARP is a nuclear protein which was used as an internal control.

The RAW 264.7 macrophages were treated with PQ in the absence or presence of LPS (0.5 µg/ml) for 6 h. Total RNA was isolated using TRIzol™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the instructions provided by the manufacturer, and the reverse transcription reaction was performed using a kit producing cDNA (Qiagen, Hilden, Germany). Polymerase chain reactions were performed using specific forward and reverse primers as follows: MCP-1 forward, 5′-AGGTCCCTGTCATGCTTCTG-3′ and reserve, 5′-TCTGGACCCATTCCTTCTTG-3′; HO-1 forward, 5′-TGAAGGAGGCCACCAAGGAGG-3′ and reverse, 5′-AGAGGTCACCCAGGTAGCGGG-3′; and β-actin forward, 5′-TGTTTG AGACCTTCAACACC-3′ and reserve, 5′-CGCTCATTGCCGATAGTGAT-3′. Parallel PCR analysis was run for the housekeeping gene, β-actin, to normalize the data for differences in mRNA quantity and integrity. The PCR products were fractionated by 1.5% agarose gel electrophoresis and stained with 5 µg/ml ethidium bromide. These experiments were performed in triplicate.

Lung tissues were obtained to evaluate the effects of PQ at 24 h after LPS injection. Mice were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co., Seoul, Korea). The lung tissues were washed and fixed in 4% (v/v) paraformaldehyde. The lung tissues were embedded in paraffin, sectioned at 4 µm thickness, and stained with an H&E solution (Sigma-Aldrich) to estimate inflammation in peribronchial and alveolar lesions. Quantitative analysis of inflammation was performed using an image analyzer (Molecular Devices).

The viability of the RAW 264.7 macrophages was examined following the treatment of the cells with various concentrations (0, 2.5, 5, 10 and 20 µg/ml) of PQ and LPS (0.5 µg/ml). Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded at 1×10⁴ cells per well of a96-well plate and incubated with PQ at various concentrations for 24 h at 37°C. After incubation, MTT (0.5 mg/ml in PBS) was added to each well, and the cells were incubated for 2 h at 37°C and 5% CO₂. The resulting formazan crystals were dissolved in DMSO. The absorbance was determined at 540 nm. The results were expressed as a percentage of surviving cells over control cells.

The data are expressed as the means ± standard deviation (SD). The statistical significance was determined using analysis of variance (ANOVA) followed by a multiple comparison test with Dunnett's adjustment. A value of p<;0.05 was considered to indicate a statistically significant diffence.

The mice with LPS-induced ALI exhibited a significant increase in the number of inflammatory cells, including neutrophils and macrophages in BALF compared with the normal control mice (Fig. 1). In particular, the number of neutrophils and macrophages in BALF was markedly increased in the mice with LPS-induced ALI. However, the PQ-treated mice exhibited a marked decrease in the number of neutrophils and macrophages in BALF compared with the mice with LPS-induced ALI in a concentration-dependent manner. Treatment with DEX was used as a positive control. The mice treated with DEX also exhibited a decrease in the number of neutrophils and macrophages in BALF compared with the LPS-exposed mice. The effects of DEX were similar to those of treatment with PQ at 15 mg/kg.

Given the fact that ALI is involved in neutrophil migration, the release of ROS and cytokine production (12), the levels of ROS and cytokines were examined in BALF. The levels of ROS were significantly increased in the LPS-exposed mice compared with the normal control mice (Fig. 2A). The mice treated with PQ at a dose of 15 mg/kg exhibited a significant reduction in ROS production compared with the LPS-exposed mice. Similar to the results obtained for ROS, the LPS-exposed mice exhibited a marked increase in the levels of TNF-α and IL-6, compared with the normal control mice, and the PQ-treated mice exhibited significantly decreased levels of TNF-α and IL-6 compared with the LPS-exposed mice (Fig. 2B and C). To further examine the effects of PQ on the production of pro-inflammatory cytokines (27), the levels of IL-6 were detected using ELISA in serum of mice. As shown Fig. 2D, PQ significantly suppressed the release of IL-6 compared with the LPS-exposed mice. The mice treated with DEX also exhibited a decrease in the levels of ROS, TNF-α and IL-6 compared with the LPS-exposed mice. The effects of DEX were similar to those of treatment with PQ at 15 mg/kg.

The infiltration of inflammatory immune cells into the lungs is one of the common characteristics of ALI. As shown by histological analysis, the LPS-exposed mice exhibited extensive infiltration of inflammatory cells into the lung tissue (Fig. 3A and B). However, the PQ-treated mice exhibited a marked reduction in inflammatory cell infiltration induced by the LPS challenge. The mice treated with DEX also exhibited a decrease in airway inflamamation compared with the LPS-exposed mice. Again, the effects of DEX were similar to those of treatment with PQ at 15 mg/kg.

iNOS has been shown to be involved in the pathogenesis of ALI (28). HO-1 inhibits the LPS-induced production of iNOS and pro-inflammatory cytokines, such as TNF-α and IL-6 (17). In this study, to examine the effects of PQ on LPS-induced ALI in mice, the expression of iNOS and HO-1 was detected using western blot analysis. As shown in Fig. 4A, the expression of iNOS was significantly increased in the lungs of mice with LPS-induced ALI. However, treatment with PQ significantly decreased iNOS expression compared with the LPS-exposed mice. In addition, PQ significantly increased the expression of HO-1 in the lungs of mice with LPS-induced ALI (Fig. 4B). Treatment with DEX also decreased the expression of iNOS and increased the expression of HO-1 in the lungs of mice compared with the LPS-exposed mice. The effects of DEX were similar to those of treatment with PQ (15 mg/kg).

MAPKs are involved in the inflammatory response in ALI (2). It is also well known that LPS administration leads to the increased phosphorylation of p38, ERK and JNK in the lungs (17). In the present study, exposure to LPS significantly increased the phosphorylation of p38, ERK and JNK in the lungs of mice. However, treatment with PQ significantly decreased the LPS-induced MAPK phosphorylation compared with the mice with LPS-induced ALI (Fig. 5). Treatment with DEX also suppressed the activation of MAPKs in the lungs of mice compared with the LPS-exposed mice. Treatment with DEX was slightly more effective in reducing MAPK phosphorylation than treatment with PQ (p<;0.01 as opposed to p<;0.05).

It is well known that the degradation of IκB and the phosphorylation of NF-κB p65 induces the transcription of most pro-inflammatory cytokines, including TNF-α and IL-6, thus playing a pivotal role in the pathogenesis of ALI (17). Thus, in the present study, to determine whether PQ affects the LPS-induced degradation of IκB and the phosphorylation of NF-κB p65, the levels of IκB and the phosphorylation of NF-κB were examined by western blot analysis. As shown in Fig. 6, the LPS administration induced the degradation of IκB and the phosphorylation of NF-κB p65. However, treatment with PQ significantly decreased the LPS-induced IκB degradation. In addition, PQ significantly decreased the phosphorylation of NF-κB p65 in the lungs of mice with LPS-induced ALI. Treatment with DEX also significantly suppressed the degradation of IκB and decreased the phosphorylation of NF-κB p65 in the lungs of mice compared to the LPS-exposed mice. The effects of DEX were similar to those of treatment with PQ (15 mg/kg).

TNF-α and IL-6 are major pro-inflammatory cytokines involved in the recruitment of neutrophils into the lungs of mice with LPS-induced ALI (17). MCP-1 is one of the key chemokines (29) that contributes to the recruitment of monocytes/macrophage into sites of immune response (30). NF-κB is a major transcription factor that is a predominant regulator of numerous pro-inflammatory cytokines and mediators, such as TNF-α, IL-6, iNOS and MCP-1 (31). We previously demonstrated that PQ attenuated the increase in iNOS protein expression in LPS-stimulated RAW 264.7 macrophages (26). In the present study, treatment with PQ inhibited the release of TNF-α and IL-6 from LPS-stimulated RAW 264.7 macrophages (Fig. 7A and B). Treatment with PQ also resulted in the suppression of NF-κB nuclear translocation, as well as in a decrease in MCP-1 expression in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner (Fig. 7C and D). No noticeable cell death was observed following treatment with PQ at the concentration of up to 20 µg/ml (Fig. 7E).

Nrf2 is a pivotal transcription factor that regulates a variety of cytoprotective enzymes, including HO-1 (32). HO-1 is an antioxidant enzyme that can be induced by stimulants, such as inflammatory oxidants and cytokines, and the induction of HO-1 attenuates ALI (33). Therefore, in this study, we used western blot analysis to determine whether PQ promotes the nuclear translocation of Nrf2 in RAW 264.7 macrophages. As shown in Fig. 8A, treatment with PQ significantly increased the nuclear translocation of Nrf2 in the RAW 264.7 cells in a concentration-dependent manner. To examine whether PQ upregulates HO-1 expression, the protein and mRNA levels of HO-1 were examined by western blot analysis and RT-PCR. Treatment with PQ resulted in a significant increase in the protein and mRNA expression of HO-1 in the RAW 264.7 macrophages in a concentration-dependent manner (Fig. 8B and C). In accordance with the increased expression of HO-1, PQ also significantly upregulated the protein and mRNA expression of HO-1 in the LPS-stimulated RAW 264.7 macrophages in a concentration-dependent manner (Fig. 8D and E).

To determine whether PQ affects the activation of MAPKs in LPS-stimulated RAW 264.7 cells, the phosphorylation levels of MAPKs were measured by western blot analysis. Exposure to LPS markedly increased the phosphorylation of MAPKs in the RAW 264.7 cells. However, treatment with PQ significantly decreased the phosphorylation of p38 MAPK in the LPS-stimulated RAW 264.7 cells (Fig. 9).