Effect of farrerol (20 and 40 mg/kg) on the IL-4, IL-5, and IL-13, Akt, P70S6K, and IκBα phosphorylation and degradation in vitro.

<p>The supernatant of Th2 cells was measured by sandwich ELISA. The values represent the mean±SEM of three independent in vitro experiments. Th2 cells were cultured with anti-CD3 (5 µg/ml) for 1 h (1 mg/L), total cellular proteins were analyzed by western blot with specific antibodies. β-Actin was used as an internal control. Experiments were repeated three times and similar results were obtained.</p

Effects of farrerol on OVA-induced AHR.

<p>Airway responsiveness of mechanically ventilated mice in response to aerosolized methacholine was measured 24 h after the last saline aerosol or OVA aerosol with pretreatment of either DMSO or Farrerol (20 and 40 mg/kg). AHR is expressed as percentage change from the baseline level of (A) dynamic compliance (Cdyn, n = 6 mice) and (B) lung resistance (RL, n = 5 mice). Cdyn refers to the distensibility of the lung and is defined as the change in volume of the lung produced by a change in pressure across the lung. RL is defined as the pressure driving respiration divided by flow. *p<0.05, **p<0.01 vs. OVA.</p

Effect of farrerol on the activation of NF-κB and IκBα phosphorylation and degradation in vivo.

<p>(A) Effect of farrerol treatment on nuclear translocation of NF-κB. Nuclear and cytoplasmic proteins from lung were analyzed by western blot with specific antibodies. (B) Effect of oxytetracycline treatment on IκBα phosphorylation and degradation. Total cellular proteins from lung were analyzed by western blot with specific antibodies. β-Actin was used as an internal control. Experiments were repeated three times and similar results were obtained.</p

Effects of farrerol on OVA-induced inflammatory cell recruitment and mucus hyper-secretion.

<p>Inflammatory cell counts in BALF obtained from sensitized mice 24 h after the last farrerol treatment. Differential cell counts were identified eosinophil (Eos), macrophage (Mac), neutrophil (Neu) and lymphocyte (Lym).</p

Effect of farrerol on Akt, P70S6K, and MAPK activation in vivo.

<p>Immunoblotting of Akt, P70S6K, and MAPK in proteins extracts of lung tissues isolated from mice 24 hours after the last OVA challenge pretreated with 20 or 40 mg/kg farrerol. β-actin was used as an internal control. Experiments were repeated three times and similar results were obtained.</p

Effects of farrerol on cytokine and chemokine levels in BALF and serum immunoglobulin production in vivo.

<p>BALF and blood were collected and centrifuged 24 hours after the last OVA challenge, and the supernatants and serum were measured by ELISA. Results of IgE in serum (mean±SEM n = 10) are expressed as Optical Density values and are representative of at least three independent in vivo experiments. *p<0.05, **p<0.01 vs. OVA.</p

Effects of farrerol on lung tissue eosinophilia and mucus production.

<p>Histologic examination of lung tissue eosinophilia using HE staining (A) and mucus secretion using AB-PAS staining (B) from: (a) PBS-challenged mice; (b) OVA-challenged mice; (c) OVA-challenged mice treated with farrerol (20 mg/kg); (d) OVA-challenged mice treated with farrerol (40 mg/kg); (e) OVA-challenged mice treated with dexamethasone (2 mg/kg, magnification×400). Quantitative analyses of inflammatory cell infiltration (C) and mucus production (D) in lung sections were performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034634#pone.0034634-Zhou2" target="_blank">[35]</a>. At least 5 different fields for each lung section was performed to score the inflammatory cells and goblet cells. Mean scores were obtained from 5 mice. **P < 0.01. vs. OVA.</p

Chemical structure of farrerol.

<p>Chemical structure of farrerol.</p

Primer sets for reverse transcriptase-polymerase chain reacion analysis.

<p>Primer sets for reverse transcriptase-polymerase chain reacion analysis.</p

Effect of farrerol on OVA-induced inflammatory gene expression in allergic airway inflammation.

<p>Lung tissues were collected 24 hours after the last OVA aerosol challenge. The mRNA in whole-lung extracts was measured by real-time PCR. Treatment with farrerol (20 and 40 mg/kg) reduced OVA-induced mRNA expression in the lung. The values represent the mean±SEM of 10 animals in each group. **P<0.01. vs. OVA.</p