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Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-?1 by Blocking TGF-?1 Binding to T?RII.

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-?1 (TGF-?1)-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-? receptor type I kinase inhibitor SB431542 and then stimulated with TGF-?1. 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 ?-smooth muscle actin (?-SMA), collagen I ? 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-? type II receptor (T?RII), and TGF-? type I receptor were detected by western blotting. T?RII knockdown with siRNA interfered with the inhibitory effect of HSYA on ?-SMA, COL1A1, and FN expression, and TGF-?1-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-?1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-?1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-?1 to MRC-5 cell cytoplasmic receptors. Also HSYA inhibited TGF-?1-activated cell expression of ?-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting T?RII in MRC-5 cells. These findings suggest that T?RII might be the target responsible for the inhibitory effects of HSYA on TGF-?1-induced pathological changes in pulmonary fibrosis.

SUBMITTER: Pan R

PROVIDER: S-EPMC5425600 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-β1 by Blocking TGF-β1 Binding to TβRII.

Pan Ruiyan R Zhang Yadan Y Zheng Meng M Zang Baoxia B Jin Ming M

Frontiers in pharmacology 20170511

Hydroxysafflor yellow A (HSYA) is an active ingredient of <i>Carthamus tinctorius</i> 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 ki ...[more]

PMID: 28553231

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Project description:ObjectiveRenal fibrosis is the common pathological foundation of many chronic kidney diseases (CKDs). The aim of this study was to investigate whether Hydroxysafflor yellow A (HSYA) can preserve renal function by inhibiting the progression of renal fibrosis and the potential mechanisms.MethodsRenal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on 7-week-old C57BL/6 mice. HSYA (10, 50 and 100 mg/kg) were intragastrically administered. Sham group and model group were administered with the same volume of vehicle. Serum and kidney samples were collected 14 days after the UUO surgery. Serum biochemical indicators were measured by automatic biochemical analyzer. Histological changes were evaluated by HE and Masson staining. In vitro, the anti-fibrotic effect of HSYA was tested on human recombinant transforming growth factor-?1 (TGF-?1) stimulated HK-2 cells. The protein levels of ?-SMA, collagen-I and fibronectin in kidney tissue and HK-2 cells were measured by immunohistochemistry and immunofluorescence. The protein levels of apoptosis-relative and TGF-?1/Smad3 signaling were detected by western blot.ResultsHSYA slowed the development of renal fibrosis both in vivo and in vitro. In UUO rats, renal function index suggested that HSYA treatment decreased the level of serum creatinine (Scr) and blood urea nitrogen (BUN) rose by UUO (P<0.05). HE staining and Masson staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration were notably attenuated in the high-dose HSYA group compared with the model group. The expressions of ?-SMA, collagen-I and fibronectin were decreased in the UUO kidney and HK-2 cells of the HSYA-treatment group. Moreover, HSYA reduced the apoptotic rate of HK-2 cells stimulated by TGF-?1. Further study revealed that HSYA regulated the TGF-?1/Smads signaling pathway both in kidney tissue and HK-2 cells.ConclusionsThese results suggested that HSYA had a protective effect against fibrosis in renal cells, at least partly, through inhibiting TGF-?1/smad3-mediated Epithelial-mesenchymal transition signaling pathway.

Project description:Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Dataset Information

Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-?1 by Blocking TGF-?1 Binding to T?RII.

Publications

Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-β1 by Blocking TGF-β1 Binding to TβRII.

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