Antifibrotic effects of eupatilin on TGF-β1-treated human vocal fold fibroblasts

Abstract

Vocal fold scarring is a major cause of dysphonia. Vocal fold fibroblasts (VFFs) and the TGF-β signaling pathway play important roles in scar formation. Eupatilin, a chromone derivative of the Artemisia species, is a traditional folk remedy for wound healing. However, until recently, few studies investigated the therapeutic effects of eupatilin. We investigated the antifibrogenic effects of eupatilin on TGF-β1-treated human vocal fold fibroblasts (hVFFs). The optimal concentration of eupatilin was determined by a cell viability assay. Western blotting was used to measure the expression of alpha-smooth muscle actin during myofibroblast differentiation, fibronectin (FN), collagen type I (Col I), and collagen type III (Col III) extracellular matrix proteins, and Smad2, Smad3, and p38 in the fibrotic pathway. Measurements were made before and after eupatilin treatment. Eupatilin at 100 nM was shown to be safe for use in hVFFs. TGF-β1 induced hVFFs to proliferate and differentiate into myofibroblasts and increased Col III and FN synthesis in a time- and dose-dependent manner. Eupatilin suppressed TGF-β1-induced hVFF proliferation and differentiation into myofibroblasts through the Smad and p38 signaling pathways. Furthermore, eupatilin inhibited TGF-β1-induced FN, Col I, and Col III synthesis in hVFFs. Our in vitro findings show that eupatilin effectively suppressed TGF-β1-induced fibrotic changes in hVFFs via the Smad and p38 signaling pathways. Thus, eupatilin may be considered a novel therapeutic agent for the treatment of vocal fold fibrosis.

Fig 1. Effect of eupatilin on cytotoxicity in human vocal fold fibroblasts (hVFFs).

The cells were incubated with eupatilin for the indicated times and at the indicated concentration. The viability of the hVFFs was then measured in a MTT assay. Data are representative of three independent experiments performed in triplicate and are expressed as the mean ± standard error of the mean (SEM). * p < 0.05 vs. untreated control (eupatilin = 0 nM).

Fig 2. Effect of eupatilin on hVFFs proliferation and motility induced by TGF-β1.

(A) hVFFs were treated for 48 h with TGF-β1 (10 ng/ml) plus 1, 10, or 100 nM eupatilin, or with 100 nM eupatilin alone. Cell proliferation was determined in an MTT assay. (B) Cell migration was evaluated by the wound healing assay. (C) Uncovered areas in the wound healing assays were quantified as the percentage of the original wound area. Data are representative of three independent experiments performed in triplicate and are expressed as the mean ± SEM. * p < 0.05 vs. TGF-β1 untreated control (TGF-β1 = 0 ng/ml).# p < 0.05 vs. eupatilin untreated control (eupatilin = 0 nM).

Fig 3. Effect of eupatilin on hVFF differentiation and TGF-β1-induced collagen deposition.

(A) FN, COL I, Col III and αSMA protein levels in hVFFs treated with TGF-β1 (10 ng/ml) and eupatilin for 48 h. (B) Protein levels were quantified and then normalized based on GAPDH expression. Data are representative of three independent experiments performed in triplicate and are expressed as the mean ± SEM. * p < 0.05 vs. TGF-β1 untreated control (TGF-β1 = 0 ng/ml and eupatilin = 0 nM). # p < 0.05 vs. eupatilin untreated control (TGF-β1 = 10 ng/ml and eupatilin = 0 nM). $ p < 0.05 vs. eupatilin treated control (TGF-β1 = 0 ng/ml and eupatilin = 100 nM).

Fig 4. Effect of eupatilin on the TGF-β1-induced phosphorylation of Smad2/3, and p38 in hVFFs.

(A) p-Smad2/3, and p-p38 protein levels in hVFFs treated with TGF-β1 (10 ng/ml) for 48 h in the presence or absence of SB203580 (10 μM) or eupatilin (100 nM). (B) Phosphorylation levels were quantified by densitometry and are presented as the ratio between the optical density of p-Smad2/3 and total Smad2/3, and p-p38 and total p38. Data are representative of three independent experiments performed in triplicate and are expressed as the mean ± SEM. * p < 0.05 vs. TGF-β1 untreated control (TGF-β1 = 0 ng/ml). # p < 0.05 vs. eupatilin untreated control (eupatilin = 0 nM).

Fig 5. Effect of eupatilin and p38 inhibitor SB203580 on TGF-β1- induced hVFF differentiation and collagen deposition.

(A) Protein levels of FN, Col I, Col III and αSMA in hVFFs treated for 48 h with TGF-β1 (10 ng/ml) in the presence or absence of SB203580 (10 μM) or eupatilin (100 nM). (B) Protein levels were quantified and then normalized based on GAPDH expression. Data are representative of three independent experiments performed in triplicate and are expressed as the mean ± SEM. * p < 0.05 vs. the TGF-β1 untreated control (TGF-β1 = 0 ng/ml and eupatilin = 0 nM). # p < 0.05 vs. untreated control (TGF-β1 = 10 ng/ml and eupatilin = 0 nM).