All experiments were approved by the institutional animal care and use committee of Chonbuk National University School of Medicine and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 82~23, revised 1996) as well as the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Dried fruiting bodies of P. linteus were purchased from the collection bank of Huazhong Agricultural University (Hubei, China). Active P. linteus was isolated using ethanol precipitation methods. Briefly, P. linteus was soaked in 50% ethanol, boiled at 90℃ for 4 h, and filtered through a 270-mesh sieve. The sludge from the primary extraction was then extracted twice using the same procedure and evaporated to dryness with a rotary vacuum evaporator (Yiheng Technology Ltd., Shanghai, China) at 40℃ under reduced pressure. The yield was 15% of the raw P. linteus. Chemical analysis revealed that polysaccharides are the major components of PLE, consisting of approximately 72% of the mixture and having an average molecular weight of 1300. Extract was filtered with a 0.2-µm syringe filter and adjusted with distilled water to give a stock solution in 1 mg/ml for experimental use. The solution was stored at 4℃ until use.

Specific pathogen-free, inbred, 7-week-old female BALB/c mice were purchased from Damool Science (Daejeon, Korea). The mice were maintained in an animal facility under standard laboratory conditions for 1 week before experimentation and provided water and standard chow ad libitum. Mice were immunized intraperitoneally with 10µg of OVA (chicken egg albumin, Sigma, St. Louis, MO, USA) plus 1.0 mg of aluminum hydroxide adjuvant (Imject® Alum; Pierce, Rockford, IL, USA). A booster injection of 10µg of OVA plus 1.0 mg aluminum hydroxide adjuvant was given 10 days later. From days 17 to 19, the immunized mice were challenged by exposure to an aerosol of 1% OVA in PBS for 20 min. The mice were divided into groups of seven animals each. Saline-sensitized and challenged mice were used as controls. PLE (10 mg/kg body weight [BW]) dissolved in saline was administered by oral gavage to each animal at 24-h intervals on days 21~23 beginning 1 h before each OVA provocation. An NF-κB inhibitor (BAY 11-7085, 20 mg/kg BW, Sigma) or a p38 MAPK inhibitor (SB 239063, 0.75 mg/kg BW, Sigma) dissolved in sterile DMSO-PBS was injected intraperitoneally into each animal twice on days 21 and 23. In the present study, a vehicle (0.1% v/v DMSO) had no significant effect on key characteristics of allergic asthma (AHR, inflammation, goblet cell metaplasia, and IgE) and important mediators (Th2 cytokines, eotaxin, and adhesion molecules) in an experimental murine model induced by exposure to OVA (data not shown).

AHR was measured 2 days after the last OVA challenge. Conscious, unrestrained mice were placed in a barometric plethysmographic chamber (Synol High-Tech, Beijing, China), and baseline readings were taken and averaged for 3 min. Aerosolized methacholine (Mch) in increasing concentrations (from 2.5 to 50 mg/ml) was then nebulized through an inlet of the main chamber for 3 min, and readings were taken and averaged for 3 min after each nebulization. The bronchopulmonary resistances are expressed as enhanced pauses (Penh), which were calculated as follows: (expiratory time/relaxation time-1)×(peak expiratory flow/peak inspiratory flow), according to the chamber manufacturer's protocol. The results are expressed as the percentage increase in Penh over the baseline after challenge performed with each concentration of Mch, where the baseline Penh (after saline challenge) is expressed as 100%.

Immediately after the assessment of airway responsiveness, mice were anesthetized and their tracheas were cannulated while the thorax was gently massaged. The lungs were lavaged with 0.7 ml of PBS. BAL fluid samples were collected, and the number of total cells in a 0.05-ml aliquot was counted using a hemocytometer (Baxter Diagnostics, Deerfield, IL, USA). The remaining samples were centrifuged, and the supernatants were stored at -70℃ until use for assays of total and OVA-specific IgE, TNF-α, IL-1β, IL-4, IL-5, IL-13, eotaxin, ICAM-1, and VCAM-1 levels. The cell pellets were resuspended in PBS, and cytospin preparations of the BAL cells were stained with Diff-Quik solution (International Reagents, Kobe, Japan). Cell differentials were then enumerated based on cell morphology and staining profiles. Counting was performed by an observer blind to the experimental treatments.

Total and OVA-specific IgE, TNF-α, IL-1β, IL-4, IL-5, IL-13, eotaxin, ICAM-1, and VCAM-1 levels in BAL fluids were determined using mouse ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The lower limits of detection for the cytokines were as follows (pg/ml): TNF-α, 5.1; IL-1β, 2.0; IL-4, 3.3; IL-5, 5.0; IL-13, 1.5; eotaxin, 3.0; ICAM-1, 0.017; and VCAM-1, 20.

Lungs were fixed with 10% formalin, and the tissues were embedded in paraffin. Fixed tissues were cut into sections 4-µm thick, placed on glass sides, and deparaffinized. Sections were stained with H&E and periodic acid-Schiff for light microscopic examination.

Freshly isolated lung tissues were homogenized in the presence of protease inhibitors, and protein concentrations were determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA). A 30-µg sample of protein from the lung homogenates was loaded per lane on a 12% SDS-PAGE gel for electrophoresis. The proteins were then transferred to nitrocellulose membranes. Western blot analysis was performed using polyclonal antibodies against poly (ADP-ribose) polymerase, β-actin (Santa Cruz Biochemicals, Santa Cruz, CA, USA), phosphorylated (p)-p38, or p38 (R&D Systems). The binding of antibodies was detected using an ECL detection system (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer's instructions.

Cytosolic or nuclear extractions from harvested lung tissues were performed as described previously (14). For western blot analysis, samples were processed using the procedure described above. NF-κB activation was assayed using the antibody against NF-κB p65 (Cell Signaling Technology, Danvers, MA, USA), IκB-α (R&D Systems), or p-IκB-α (Santa Cruz).

To inhibit endogenous protease activity, we added 1 mM PMSF to lung nuclear extracts. An oligonucleotide containing a κ-chain binding site (κB, 5'-CCGGTTAACAGAGGGGGCTTTCCGAG-3') was used as an EMSA probe. The two complimentary strands were annealed and labeled with [α-³²P] deoxycytidine triphosphate. Labeled probe (10,000 cpm), 10 µg of nuclear extract, and binding buffer (10 mM Tris-HCl, pH 7.6, 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng poly(deoxyinosinic-deoxycytidylic) acid, and 1 mM dithiothreitol) were then incubated for 30 min at room temperature in a final volume of 20µl. Reaction mixtures were analyzed using electrophoresis on 4% polyacrylamide gels in 0.5×Tris-borate buffer. DNA-protein interactions were specific for NF-κB as demonstrated by competition EMSA using a 50-fold excess of unlabeled oligonucleotide (data not shown).

All immunoreactive and phosphorylation signals were analyzed with densitometric scanning (Gel Doc XR; Bio-Rad, Hercules, CA, USA). Data are expressed as means±SEM. Statistical evaluation of the data was performed with ANOVA followed by Dunnett's post-hoc test using Prism 5 software (GraphPad Software, San Diego, CA, USA). Results with a p value of <0.05 were considered statistically significant.

One functional consequence of the inflammatory process that underlies asthma is AHR. In this study, AHR was determined in Penh and was substantially increased in OVA-challenged mice compared with control mice in response to Mch inhalation. PLE, SB 239063, or BAY 11-7085 dramatically prevented AHR in response to inhaled Mch, as shown in Fig. 1A, suggesting that in vivo immune-mediated pathology was modified. Next, to examine the effect of PLE on chemotaxis-that is, the recruitment of inflammatory cells into the airway-we counted inflammatory cells in BAL fluids. We analyzed the cellular composition of the BAL fluids of mice 48 h after the last OVA challenge. In saline-treated mice, OVA challenge resulted in a marked increase in eosinophils and slight increases in neutrophils and lymphocytes compared with counts in control mice (Fig. 1B). However, pretreatment with PLE, SB 239063, or BAY 11-7085 significantly attenuated OVA-induced recruitment of eosinophils (p<0.05). The observed reduction in chemotaxis in the airway correlated with the histological changes in lung parenchyma. Lungs from OVA-challenged mice showed widespread perivascular and peribronchiolar inflammatory cell infiltrates (Fig. 1C and D). Moreover, the percentage of mucus-producing goblet cells (indicated by periodic acid-Schiff staining) in OVA-challenged mice was substantially greater than that in control mice (see Fig. 1C and E). However, administration of PLE significantly reduced inflammatory cell infiltration and goblet cell hyperplasia. These results indicate that PLE efficiently attenuates allergic airway inflammation and mucus hypersecretion in OVA-induced asthmatic mice.

Total and OVA-specific IgE levels were determined with ELISA in each experimental group. IgE levels in BAL fluids were dramatically elevated in OVA-challenged mice compared with those in control mice. However, the administration of PLE or BAY 11-7085 to OVA-challenged mice significantly reduced total and OVA-specific IgE levels (Fig. 2).

Allergic asthmatic inflammation is caused by the secretion of a series of proinflammatory (TNF-α and IL-1β) and Th2 (IL-4, IL-5, and IL-13) cytokines (15). To assess the effect of PLE on pulmonary inflammation in asthmatic mice, we measured the levels of these cytokines in BAL fluids. ELISA showed that the levels of TNF-α, IL-1β, IL-4, IL-5, and IL-13 in BAL fluids were significantly increased in OVA-challenged mice compared with those in control mice (see Fig. 2). These increases were significantly decreased by the administration of PLE or BAY 11-7085. Moreover, given that chemokines and leukocyte-endothelial adhesion molecules are central in the recruitment and migration of leukocytes to the sites of inflammation (16), the levels of eotaxin, ICAM-1, and VCAM-1 were measured. Changes in the levels of these cytokines were similar to those observed for the aforementioned cytokines, indicating that OVA challenge-induced increases in cytokine levels can be reversed by PLE or BAY 11-7085.

In view of the present data that AHR and eosinophilic inflammation in asthmatic mice are blocked by BAY 11-7085, a specific NF-κB inhibitor, and that NF-κB plays a key role in allergic lung inflammation by inducing the transcription of various proinflammatory mediators (3), we hypothesized that PLE attenuates airway inflammatory reactions by suppressing NF-κB activation. To test this hypothesis, we first studied the nuclear translocation of the NF-κB p65 subunit in lung tissues after OVA challenge and observed a decrease and an increase of NF-κB p65 levels in the cytosol and nuclei, respectively, of OVA-challenged lungs (Fig. 3A). By contrast, cytosolic and nuclear extracts from PLE- or SB 239063-treated mice displayed suppressed nuclear translocation.

Next, the effects of PLE on OVA-induced phosphorylation and degradation of IκB-α were evaluated to clarify the molecular mechanisms through which PLE inhibits NF-κB activity. PLE and SB 239063 significantly reduced the OVA-induced phosphorylation and degradation of IκB-α in the cytosol of OVA-challenged lung tissues (Fig. 3B). Furthermore, EMSA revealed an increase in the binding activity of lung nuclear extracts to the NF-κB consensus sequence in OVA-exposed mice (Fig. 3C) compared with control mice, whereas PLE markedly blocked the DNA binding activity of NF-κB. Taken together, these findings indicate that PLE represses NF-κB transcriptional activity, possibly by stabilizing IκB-α and impairing the nuclear transport of the p65 subunit in the lung tissues of OVA-challenged mice.

To investigate whether the inhibition of asthmatic response by PLE is mediated through p38 MAPK, we examined the phosphorylation of p38 MAPK in OVA-challenged mice pretreated with PLE or SB 239063, a p38 MAPK inhibitor. As shown in Fig. 4, PLE and SB 239063 effectively inhibited OVA-induced phosphorylation of p38 MAPK in allergic mice. On the contrary, the expression of p38 was unaffected by OVA, PLE, or SB 239063.