Clin Exp Otorhinolaryngol.  2014 Dec;7(4):295-301. 10.3342/ceo.2014.7.4.295.

Role of Caffeic Acid on Collagen Production in Nasal Polyp-Derived Fibroblasts

• Chung SW
• Park IH
• Hong SM
• Cho JS
• Moon JH
• Kim TH
• Lee HM

• KMID: 2278370
• DOI: http://doi.org/10.3342/ceo.2014.7.4.295

Abstract

OBJECTIVES
Caffeic acids are known to have anti-oxidant, anti-inflammatory, immunomodulatory, and tissue reparative effects. The purposes of this study were to determine the effect of caffeic acid on transforming growth factor (TGF) beta1-induced myofibroblast differentiation and collagen production, and to determine whether caffeic acid is involved in the antioxidant effect in nasal polyp-derived fibroblasts (NPDFs).
METHODS
NPDFs were pretreated with caffeic acid (1-10 microM) for 2 hours and stimulated with TGF-beta1 (5 ng/mL) for 24 hours. The expression of alpha-smooth muscle actin (SMA), collagen types I and III, and Nox4 mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by actin ned by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the Sircol collagen dye-binding assay. The reactive oxygen species (ROS) generated by NPDFs were determined using 2',7'-dichlorfluorescein-diacetate. siNox4 was used to determine the effect of Nox4.
RESULTS
The expression of alpha-SMA and production of collagen were significantly increased following TGF-beta1 treatment. In contrast, the level of expression of alpha-SMA and the level of production of collagen were decreased by pretreatment with caffeic acid. The activation of Nox4 and the subsequent production of ROS were also reduced by pretreatment with caffeic acid. The expression of alpha-SMA was prevented by inhibition of ROS generation with siNox4.
CONCLUSION
Caffeic acid may inhibit TGF-beta1-induced differentiation of fibroblasts into myofibroblasts and collagen production by regulating ROS.

Keyword

Nasal polyps; Myofibroblasts; Transforming growth factor beta1; Caffeic acid; Alpha-smooth muscle actin; Collagen

MeSH Terms

Actins
Antioxidants
Caffeic Acids
Collagen*
Fibroblasts*
Microscopy, Fluorescence
Myofibroblasts
Nasal Polyps
Reactive Oxygen Species
RNA, Messenger
Transforming Growth Factor beta1
Transforming Growth Factors
Actins
Antioxidants
Caffeic Acids
Collagen
RNA, Messenger
Reactive Oxygen Species
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Fig. 1 Cytotoxicity test using 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-2H-tetrazolium bromide assay at various concentrations of caffeic acid phenethyl ester (CAPE) in nasal polyp-derived fibroblasts. Values are expressed as the mean±SE of 6 separate experiments.

• 

  Fig. 2 Effects of caffeic acid phenethyl ester (CAPE) on expression of α-smooth muscle actin (SMA) mRNA in transforming growth factor (TGF) β1-induced nasal polyp fibroblasts, as determined by RT-PCR (A) and densitometric analysis (B). The expression of GAPDH is shown as an internal control. Cells were also treated with 0.1% demethyl sulfoxide (DMSO) in the absence of CAPE. Values are expressed as the mean±standard error of six separate experiments. Effects of CAPE on α-SMA protein expression in TGF-β1-induced nasal polyp-derived fibroblasts (NPDFs), as determined by immunofluorescent staining (C). NPDFs were treated with media alone. NPDFs induced by TGF-β1. NPDFs induced by TGF-β1 were pretreated with CAPE (1 µM) and CAPE (5 µM). Original magnification (×200). *P<0.05 vs. control, †P<0.05 vs. TGF-β1 alone. Scale bar=100 µm.

• 

  Fig. 3 Effects of caffeic acid phenethyl ester (CAPE) on the expression of collagen types I and III mRNA in transforming growth factor (TGF) β1-induced NPDFs, as determined by RT-PCR (A, representative of six independent experiments) and densitometric analysis (B). The expression of GAPDH is shown as an internal control. Cells were also treated with 0.1% demethyl sulfoxide (DMSO) in the absence of CAPE. The effects of CAPE on total soluble collagen production in TGF-β1-induced nasal polyp-derived fibroblasts were determined by a Sircol collagen dye-binding assay (C). Values are expressed as the mean±SE. *P<0.05 vs. control, †P<0.05 vs. TGF-β1 alone.

• 

  Fig. 4 Fluorescent microscopic finding of intracellular reactive oxygen species (ROS), original magnification (×200). Control fibroblasts expressed small amount of ROS production. After treatment with transforming growth factor (TGF) β1, ROS markedly increased. Pretreatment with caffeic acid phenethyl ester (CAPE, 1 and 5 µM) strongly inhibited induced production of ROS in dose-dependent manner. (A) No treatment. (B) TGF-β1 (5 ng/mL) alone. (C) TGF-β1 and CAPE (1 µM). (D) TGF-β1 and CAPE (5 µM). Scale bar=100 µm.

• 

  Fig. 5 Effect of caffeic acid phenethyl ester (CAPE) on Nox4 mRNA expression in transforming growth factor (TGF) β1-induced nasal polyp-derived fibroblasts, as determined by RT-PCR (A, representative of six independent experiments) and densitometric analysis (B). The expression of GAPDH is shown as an internal control. Cells were also treated with 0.1% demethyl sulfoxide (DMSO) in the absence of CAPE. Values are presented as the mean±SE. *P<0.05 vs. control, †P<0.05 vs. TGF-β1 alone.

• 

  Fig. 6 Nox4 mediates transforming growth factor (TGF) β1-induced α-smooth muscle actin (SMA) mRNA in nasal polyp-derived fibroblasts. Cells were transfected with small interfering RNA (siCont or siNox4) and stimulated for 12 hours with TGF-β1, and α-SMA mRNA was semi-quantified by RT-PCR (A). Mean±SE of densitometric data (B). As a control, the universal negative control siRNA (siCont) were employed. *P<0.05 vs. control, †P<0.05 vs. TGF-β1 alone.

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