Allergy Asthma Immunol Res.  2017 Jan;9(1):25-34. 10.4168/aair.2017.9.1.25.

Inhibitory Effects of Resveratrol on Airway Remodeling by Transforming Growth Factor-β/Smad Signaling Pathway in Chronic Asthma Model

• Lee HY
• Kim IK
• Yoon HK
• Kwon SS
• Rhee CK
• Lee SY

• KMID: 2355891
• DOI: http://doi.org/10.4168/aair.2017.9.1.25

Abstract

PURPOSE
Asthma is a chronic airway disease characterized by airway remodeling, leading to a progressive decline in lung function. Therapeutic agents that attenuate airway remodeling can complement the limited effects of traditional glucocorticoids. In this study, we investigated the effect of resveratrol on allergic airway inflammation and remodeling in a murine model of chronic bronchial asthma.
METHODS
Peribronchial smooth muscle thickening that developed in mice challenged with a 3-month repeated exposure to ovalbumin (OVA) was used to study airway remodeling. Oral resveratrol was administered daily during the OVA challenge. The expression of TGF-β1/Smad signaling proteins and downstream mesenchymal markers in the presence or absence of resveratrol was examined in bronchial epithelial cells.
RESULTS
OVA sensitization and chronic challenge increased airway hyperresponsiveness, inflammation, goblet cell hyperplasia, α-smooth muscle actin (SMA), and collagen deposition. Resveratrol effectively suppressed OVA-induced airway inflammation and remodeling. The expression of TGF-β1/phosphorylated Smad2/3 was increased in the lung tissues of OVA-challenged mice but effectively inhibited by resveratrol. In bronchial epithelial cells, the TGF-β1-induced expression of the mesenchymal markers snail, slug, vimentin, and α-SMA was suppressed by resveratrol treatment.
CONCLUSIONS
Resveratrol effectively ameliorated both airway inflammation and airway structural changes in a mouse model of bronchial asthma. These effects were mediated by decreased TGF-β1 expression, in turn suppressing TGF-β1/Smad signaling and the epithelial-mesenchymal transition process. Our results demonstrate the potential benefits of resveratrol for the treatment of airway remodeling associated with bronchial asthma.

Keyword

Resveratrol; TGF-β1; airway remodeling

MeSH Terms

Actins
Airway Remodeling*
Animals
Asthma*
Collagen
Complement System Proteins
Epithelial Cells
Epithelial-Mesenchymal Transition
Gastropoda
Glucocorticoids
Goblet Cells
Hyperplasia
Inflammation
Lung
Mice
Muscle, Smooth
Ovalbumin
Ovum
Snails
Vimentin
Actins
Collagen
Complement System Proteins
Glucocorticoids
Ovalbumin
Vimentin

Figure

• 

  Fig. 1 Effect of resveratrol on airway hyperresponsiveness (AHR) to methacholine (Mch). AHR was measured using the flexiVent system 24 hours after the final ovalbumin (OVA) challenge. The concentration of Mch was increased from 6.25 to 50 mg/mL. OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. The values are means±SEM (n=5/group). *P<0.05 vs the OVA group.

• 

  Fig. 2 Effect of resveratrol on total and differential cell counts in bronchoalveolar lavage (BAL) fluid. BAL was performed immediately after the measurement of AHR. OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. The values are means±SEM (n=8-12/group). *P<0.05 vs the OVA group.

• 

  Fig. 3 Effects of resveratrol on cytokine levels in BAL fluid. Interleukin (IL)-4, IL-5, and IL-13 levels in the supernatant of BAL fluid of OVA-sensitized/challenged mice were measured using an ELISA. RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. The values are means±SEM (n=8-12/group). *P<0.05 vs the OVA group.

• 

  Fig. 4 Effects of resveratrol on peribronchial inflammation in lung tissue. (A) The fixed tissues were embedded in paraffin, sectioned (4-µm thick), and then stained with H&E (×200). (B) The effects of resveratrol on goblet cell hyperplasia in lung tissue. Paraffin-embedded tissues were sectioned (5- to 6-µm thick) and then stained with periodic acid-Schiff (PAS). (C) Goblet cell hyperplasia was quantified based on the percentage of goblet cells in the epithelium, scored as follows: grade 0 (no goblet cells); grade 1 (<25%); grade 2 (25%-50%); grade 3 (51%-75%); grade 4 (>75%). The mean goblet cell hyperplasia score was calculated for each mouse (n=8-12/group). OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. *P<0.05 vs the OVA group.

• 

  Fig. 5 Effects of resveratrol on area of peribronchial smooth muscle. (A) Peribronchial α-smooth muscle actin was immunostained in paraffin-embedded lung sections (red). (B) The immunostained area was quantified using an image analysis system. The results are expressed as the immunostained area per unit basement membrane length (µm) in the bronchioles of each mouse (n=8-12/group). OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. *P<0.05 vs the OVA group.

• 

  Fig. 6 Effect of resveratrol on total collagen levels in lung tissue (60 mg/mouse) determined by hydroxyproline assay. OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. The values are means±SEM (n=8-12/group). *P<0.05 vs the OVA group.

• 

  Fig. 7 Effect of resveratrol on the expression of TGF-β1 and phosphorylated (p)-Smad in BAL fluid and lung tissues. (A) TGF-β1 expression was measured in BAL fluid by ELISA. *P<0.05 vs the OVA group. (B) Quantitative real-time reverse-transcription polymerase change reaction (qRT-PCR) analysis of TGF-β1 gene in homogenized lung tissues. *P<0.05 vs the OVA group. (C and D) Expression of TGF-β1, p-Smad2, and p-Smad3 proteins in lung tissues as determined by western blotting. OVA, OVA-sensitized/challenged mice; RES (10 mg/kg), 10 mg resveratrol/kg+OVA; RES (50 mg/kg), 50 mg resveratrol/kg administered to sensitized/challenged mice. The values are means±SEM (n=8-12/group). *P<0.05 and †P<0.01 vs the control group, ‡P<0.05 and §P<0.01 vs the OVA group, ∥P<0.01 vs the OVA+RES10 group.

• 

  Fig. 8 Effects of resveratrol on the expression of epithelial-mesenchymal transition (EMT) protein markers and Smad proteins in BEAS-2B cells treated for 48 hours with 5 µM TGF-β1 with or without 10 or 20 µM of resveratrol. (A) The expression of snail and slug mRNA was measured by quantitative reverse transcription (qRT)-PCR. (B) Mesenchymal markers (slug, snail and α-smooth muscle actin) were examined by western blot analysis. (C) The expression of Smad2, p-Smad2 and Smad4 proteins were determined by western blotting. CON, control; TGFβ, TGF-β1 (5 µM) treated only; Res10, treated with 10 µM resveratrol; Res20, 20 µM resveratrol; TGFβ+Res10, TGF-β1 and resveratrol 10 µM; TGFβ+Res20, TGF-β1 and resveratrol 20 µM. *P<0.05 and †P<0.01 vs the TGF-β1-treated cells. ‡P<0.05 vs TGF-β+resveratrol (10 µM)-treated cells.

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