Allergy Asthma Immunol Res.  2015 May;7(3):276-282. 10.4168/aair.2015.7.3.276.

Inhibitory Effect of Delphinidin on Extracellular Matrix Production via the MAPK/NF-kappaB Pathway in Nasal Polyp-Derived Fibroblasts

Affiliations
  • 1Brain Korea 21 Plus for Biomedical Science, Korea University, College of Medicine, Seoul, Korea. lhman@korea.ac.kr
  • 2Institute for Medical Devices Clinical Trial Center, Korea University, College of Medicine, Seoul, Korea.
  • 3Guro Hospital Department of Otorhinolaryngology Head and Neck Surgery, Korea University, College of Medicine, Seoul, Korea.

Abstract

PURPOSE
Nasal polyps are associated with chronic inflammation of the mucous membranes in the nose and paranasal sinuses and involved in extracellular matrix (ECM) accumulation. Delphinidin promotes ECM degradation in hepatitis and cardiac fibrosis. The aims of this study were to examine the inhibitory effect of delphinidin on TGF-beta1-induced myofibroblast differentiation and ECM accumulation, and to determine the underlying mechanisms in nasal polyp-derived fibroblasts (NPDFs).
METHODS
NPDFs were stimulated with TGF-beta1, with or without delphinidin, and the expression levels of alpha-SMA, fibronectin, and collagen type I were determined by RT-PCR, Western blot analysis, and collagen assay. The expression of alpha-SMA protein was measured by immunocytochemical staining. Mitogen-activated protein kinase and NF-kappaB activation induced by TGF-beta1 were determined by Western blot analysis. The transcriptional activity of NF-kappaB was measured by luciferase assay.
RESULTS
The expression levels of alpha-SMA, fibronectin, and collagen type I increased in TGF-beta1-stimulated NPDFs. In TGF-beta1-induced NPDFs, delphinidin inhibited the expression of alpha-SMA, fibronectin, and collagen. Inhibitors of MAPK and NF-kappaB blocked the expression of alpha-SMA, fibronectin, and collagen type I. Delphinidin suppressed the activation of MAPK and NF-kappaB induced by TGF-beta1 stimulation.
CONCLUSIONS
These results suggest that delphinidin may inhibit TGF-beta1-induced myofibroblast differentiation and ECM production through the MAPK/NF-kappaB signaling pathway in NPDFs.

Keyword

Chronic rhinosinusitis; nasal polyposis; TGF-beta1; extracellular matrix; myofibroblast; delphinidin; MAPK; NF-kappaB

MeSH Terms

Blotting, Western
Collagen
Collagen Type I
Extracellular Matrix*
Fibroblasts*
Fibronectins
Fibrosis
Hepatitis
Inflammation
Luciferases
Mucous Membrane
Myofibroblasts
Nasal Polyps
NF-kappa B
Nose
Paranasal Sinuses
Protein Kinases
Transforming Growth Factor beta1
Collagen
Collagen Type I
Fibronectins
Luciferases
NF-kappa B
Protein Kinases
Transforming Growth Factor beta1

Figure

  • Fig. 1 Effect of delphinidin on myofibroblast differentiation (α-SMA) induced by TGF-β1 in NPDFs. NPDFs were treated with TGF-β1 (5 ng/mL), with or without delphinidin (Del, 0-20 µM). The mRNA expression level of α-SMA was determined by RT-PCR (A). Protein production of α-SMA was measured by Western blot (B) and visualized by immunofluorescence staining with an antibody to α-SMA (C). Values are the means ±SEM of the 3 independent experiments. Images were acquired by confocal laser scanning microscopy. *P<0.05 vs control; †P<0.05 vs TGF-β1. Scale bar=50 µm.

  • Fig. 2 Effect of delphinidin on extracellular matrix production induced by TGF-β1 in NPDFs. NPDFs were treated with TGF-β1 (5 ng/mL), with or without delphinidin (Del, 0-20 µM). The mRNA expression levels of collagen type I and fibronectin were determined by RT-PCR (A). The protein expression level of fibronectin was measured by Western blot (B), and the total amount of collagen was measured by Sircol assay (C). Values are the means ±SEM of the 3 independent experiments. *P<0.05 vs control; †P<0.05 vs TGF-β1.

  • Fig. 3 Effect of delphinidin on TGFβ1-induced MAPK signaling pathways in NPDFs. NPDFs were stimulated with TGF-β1 (5 ng/mL), with or without delphinidin (20 µM) and MAPK inhibitors, including those for ERK (U0126, 10 µM), p38 (SB203580, 10 µM), and JNK (SP600125, 10 µM). (A) The phosphorylation of MAPK signaling molecules, including p-ERK, p-JNK, and p-p38, was measured by Western blot. (B) The protein expression levels of α-SMA and fibronectin were determined by Western blot. (C) The total amount of collagen was measured by collagen assay. Values are the means ±SEM of the 3 independent experiments. *P<0.05 vs control; †P<0.05 vs TGF-β1.

  • Fig. 4 Effect of delphinidin on TGFβ1-induced NF-κB signaling in NPDFs. NPDFs were stimulated with TGF-β1 (5 ng/mL), with or without delphinidin (Del, 20 µM), NF-κB inhibitor (BAY-11, 1 µM) or MAPK inhibitors. (A) p50 protein production was determined by Western blot. (B) The protein expressions of Fibronectin and α-SMA were examined by Western blot. (C) The total amount of collagen was measured by Sircol assay. (D) The transcriptional activity of NF-κB was measured by luciferase assay. Values are the means ±SEM of the 3 independent experiments. *P<0.05 vs control; †P<0.05 vs TGF-β1.

  • Fig. 5 Schematic diagram of the role of delphinidin in TGF-β1-induced myofibroblast differentiation and extracellular matrix production in NPDFs.


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