Abstract
Hydroxysafflor yellow A (HSYA) is an active ingredient of Carthamus tinctorius L.. This study aimed to evaluate the effects of HSYA on transforming growth factor-β1 (TGF-β1)-induced changes in proliferation, migration, differentiation, and extracellular matrix accumulation and degradation in human fetal lung fibroblasts (MRC-5), to explore the mechanisms whereby HSYA may alleviate pulmonary fibrosis. MRC-5 cells were incubated with various doses of HSYA and/or the TGF-β receptor type I kinase inhibitor SB431542 and then stimulated with TGF-β1. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt assay. Cell migration was detected by wound-healing assay. Protein levels of α-smooth muscle actin (α-SMA), collagen I α 1 (COL1A1), and fibronectin (FN) were measured by immunofluorescence. Protein levels of matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-2, TGF-β type II receptor (TβRII), and TGF-β type I receptor were detected by western blotting. TβRII knockdown with siRNA interfered with the inhibitory effect of HSYA on α-SMA, COL1A1, and FN expression, and TGF-β1-induced Sma and Mad protein (Smad), and extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway activation. The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-β1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-β1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-β1 to MRC-5 cell cytoplasmic receptors. Also HSYA inhibited TGF-β1-activated cell expression of α-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting TβRII in MRC-5 cells. These findings suggest that TβRII might be the target responsible for the inhibitory effects of HSYA on TGF-β1-induced pathological changes in pulmonary fibrosis.
Highlights
MATERIALS AND METHODSIdiopathic pulmonary fibrosis (IPF) is defined as chronic, unrelenting, and progressive fibrosing interstitial pneumonia with unknown cause (Raghu et al, 2011; Puglisi et al, 2016)
The optical density (OD) values for the transforming growth factor-β1 (TGF-β1)+SB431542 treated cells were comparable to those of the TGF-β1+SB431542+Hydroxysafflor yellow A (HSYA) treated group, suggesting that the effect of HSYA was dependent on the Transforming growth factor-β (TGF-β) receptor type I kinase pathway
Cell migration was similar in the TGF-β1+SB431542 and TGF-β1+SB431542+HSYA groups, suggesting that the effect of HSYA was dependent on the TGF-β receptor type I kinase pathway
Summary
Idiopathic pulmonary fibrosis (IPF) is defined as chronic, unrelenting, and progressive fibrosing interstitial pneumonia with unknown cause (Raghu et al, 2011; Puglisi et al, 2016). Increasing evidence indicates that IPF is caused by the abnormal behavior of alveolar epithelial cells, which provoke fibroblast proliferation, migration, and differentiation. Activated fibroblasts secrete increased extracellular matrix (ECM) molecules, resulting in subsequent destruction of the lung architecture (King et al, 2011). It is considered to be a crucial molecule involved in the activation of the fibrosis (Leask and Abraham, 2004). Active levels of TGF-β1 were reportedly increased in the lungs of IPF patients and in a bleomycin (BLM)-induced pulmonary fibrosis model (Coker et al, 1997, 2001; Shimbori et al, 2013; Wolters et al, 2014). Inhibitors of the TGF-β1 signaling pathway have emerged as potential therapies for IPF
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