Lab Anim Res.  2017 Jun;33(2):76-83. 10.5625/lar.2017.33.2.76.

Pine bark extract (Pycnogenol®) suppresses cigarette smoke-induced fibrotic response via transforming growth factor-β1/Smad family member 2/3 signaling

• Ko JW
• Shin NR
• Park SH
• Kim JS
• Cho YK
• Kim JC
• Shin IS
• Shin DH

• KMID: 2407419
• DOI: http://doi.org/10.5625/lar.2017.33.2.76

Abstract

Chronic obstructive pulmonary diseases (COPD) is an important disease featured as intense inflammation, protease imbalance, and air flow limitation and mainly induced by cigarette smoke (CS). In present study, we explored the effects of Pycnogenol® (PYC, pine bark extract) on pulmonary fibrosis caused by CS+lipopolysaccharide (LPS) exposure. Mice were treated with LPS intranasally on day 12 and 26, followed by CS exposure for 1 h/day (8 cigarettes per day) for 4 weeks. One hour before CS exposure, 10 and 20 mg/kg of PYC were administered by oral gavage for 4 weeks. PYC effectively reduced the number of inflammatory cells and proinflammatory mediators caused by CS+LPS exposure in bronchoalveolar lavage fluid. PYC inhibited the collagen deposition on lung tissue caused by CS+LPS exposure, as evidenced by Masson's trichrome stain. Furthermore, transforming growth factor-β1 (TGF-β1) expression and Smad family member 2/3 (Smad 2/3) phosphorylation were effectively suppressed by PYC treatment. PYC markedly reduced the collagen deposition caused by CS+LPS exposure, which was closely involved in TGF-β1/Smad 2/3 signaling, which is associated with pulmonary fibrotic change. These findings suggest that treatment with PYC could be a therapeutic strategy for controlling COPD progression.

Keyword

Pycnogenol; chronic obstructive pulmonary disease; cigarette smoke; collagen deposition; transforming growth factor-β1/Smad family member 2/3

MeSH Terms

Animals
Bronchoalveolar Lavage Fluid
Collagen
Humans
Inflammation
Lung
Lung Diseases, Obstructive
Mice
Phosphorylation
Pulmonary Disease, Chronic Obstructive
Pulmonary Fibrosis
Smoke
Tobacco Products*
Collagen
Smoke

Figure

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  Figure 1 PYC treatment inhibited the inflammatory cell counts in the mouse BALF. Cells were isolated by centrifugation and stained with the Diff-Quik staining reagent. NC, normal control mice; CS+LPS, CS- and LPS-induced mice; ROF, CS- and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P<0.01; *,**Significantly different from CS+LPS, P<0.05, P<0.01, respectively.

• 

  Figure 2 PYC treatment inhibited proinflammatory cytokine levels in the mouse BALF. IL-1β, IL-6, and TNF-α levels were determined by ELISA. NC, normal control mice; CS+LPS, CS- and LPS-induced mice; ROF, CS- and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P<0.01; **Significantly different from CS+LPS, P<0.01.

• 

  Figure 3 PYC treatment inhibited airway inflammation. Histological examination of airway inflammation, performed in lung tissue using H&E staining. NC, normal control mice; CS+LPS, CS- and LPS-induced mice; ROF, CS- and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P<0.01; *,**Significantly different from CS+LPS, P<0.05, P<0.01, respectively.

• 

  Figure 4 PYC treatment inhibited collagen deposition. Histological examination of collagen deposition (blue color), performed in lung tissue using Masson's trichrome staining. NC, normal control mice; CS+LPS, CS- and LPS-induced mice; ROF, CS- and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P< 0.01; *,**Significantly different from CS+LPS, P<0.05, P< 0.01, respectively.

• 

  Figure 5 PYC treatment inhibited TGF-β1 and collagen expression levels in lung tissue of mice. (A) Immunohistochemical analysis for TGF-β1 positivity performed in lung tissue. (B) Immunohistochemical analysis for collagen positivity performed in lung tissue. NC, normal control mice; CS+LPS: CS- and LPS-induced mice; ROF, CS and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P<0.01; **Significantly different from CS+LPS, P<0.01.

• 

  Figure 6 PYC reduced TGF-β1 and collagen protein levels and Smad 2/3 phosphorylation in lung tissue. Protein expression was assessed by western blot analysis and relative ratio of protein expression was evaluated. β-Actin was used as a protein loading control. NC, normal control mice; CS+LPS, CS- and LPS-induced mice; ROF, CS- and LPS-induced mice treated with roflumilast (10 mg/kg); PYC-10, CS- and LPS-induced mice treated with PYC (10 mg/kg); PYC-20, CS- and LPS-induced mice treated with PYC (20 mg/kg). The values are expressed as the means±SD (n=7). ##Significantly different from NC, P<0.01; *,**Significantly different from CS+LPS, P<0.05, P<0.01, respectively.

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