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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.

SUBMITTER: Park SJ

PROVIDER: S-EPMC7993872 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100 mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5 mM 2-O-α-d-glucopyranosyl-l-ascorbic acid (VitC) ± 2 ng/ml of T1 or T3. After 4 h or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 h, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

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

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