Sulforaphane attenuates pulmonary fibrosis by inhibiting the epithelial-mesenchymal transition

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective treatment. The epithelial-mesenchymal transition (EMT) is a critical stage during the development of fibrosis. To assess the effect of sulforaphane (SFN) on the EMT and fibrosis using an in vitro transforming growth factor (TGF)-β1-induced model and an in vivo bleomycin (BLM)-induced model.

Methods: In vitro studies, cell viability, and cytotoxicity were measured using a Cell Counting Kit-8. The functional TGF-β1-induced EMT and fibrosis were assessed using western blotting and a quantitative real-time polymerase chain reaction. The lungs were analyzed histopathologically in vivo using hematoxylin and eosin and Masson's trichrome staining. The BLM-induced fibrosis was characterized by western blotting and immunohistochemical analyses for fibronectin, TGF-β1, E-cadherin (E-cad), and α-smooth muscle actin (SMA) in lung tissues.

Results: SFN reversed mesenchymal-like changes induced by TGF-β1 and restored cells to their epithelial-like morphology. The results confirmed that the expression of the epithelial marker, E-cadherin, increased after SFN treatment, while expression of the mesenchymal markers, N-cadherin, vimentin, and α-SMA decreased in A549 cells after SFN treatment. In addition, SFN inhibited TGF-β1-induced mRNA expression of the EMT-related transcription factors, Slug, Snail, and Twist. The SFN treatment attenuated TGF-β1-induced expression of fibrosis-related proteins, such as fibronection, collagen I, collagen IV, and α-SMA in MRC-5 cells. Furthermore, SFN reduced the TGF-β1-induced phosphorylation of SMAD2/3 protein in A549 cells and MRC-5 cells. BLM induced fibrosis in mouse lungs that was also attenuated by SFN treatment, and SFN treatment decreased BLM-induced fibronectin expression, TGF-β1 expression, and the levels of collagen I in the lungs of mice.

Conclusions: SFN showed a significant anti-fibrotic effect in TGF-β-treated cell lines and BLM-induced fibrosis in mice. These findings showed that SFN has anti-fibrotic activity that may be considered in the treatment of IPF.

Keywords: Bleomycin; Epithelial-mesenchymal transition; Idiopathic pulmonary fibrosis; Sulforaphane.

Fig. 1

The effect of sulphoraphane (SFN) treatment on the morphology and protein markers in the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in A549 epithelial cells. The cells were pre-treated with the indicated concentrations of SFN for 1 h and then stimulated with TGF-β1 (1.0 ng/ml) for 24 h except α-smooth muscle actin for 72 h. The effects of SFN on cell viability for 24, 48, and 72 h (b). SFN treatment restored the TGF-β1-induced changes in epithelial morphology with original magnification, × 200 (a). Western blot analysis of the epithelial cell marker (E-cadherin), and the mesenchymal markers (N-cadherin, vimentin, and α-smooth muscle actin) (c). The mRNA levels of EMT-related transcription factors including Slug (d), Snail (e), and Twist (f) were measured by the quantitative real-time polymerase chain reaction (qRT-PCR). The data are expressed as means ± standard deviation of at least three different experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 versus the control; ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus TGF-β1 induction

Fig. 2

The effect of SFN on the expression of protein markers in TGF-β1-induced fibrosis in MRC-5 fibroblast cells. The cells were pre-treated for 1 h with the indicated concentrations of SFN and for 48 h with TGF-β1 (5.0 ng/ml). The effects of SFN on cell viability for 24, 48, and 72 h (a). The protein levels of fibronectin, collagen, and α- SMA were measured by western blot analyses (b). The mRNA levels of fibronectin (c), type I collagen (d), type IV collagen (e) and α-SMA (f) were measured by the qRT-PCR. The data are expressed as means ± the standard deviation of at least three different experiments. ^**p < 0.01 versus the control; ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 versus TGF-β1 induction

Fig. 3

The effect of SFN on TGF-β/SMAD signalling in A549 epithelial cells and MRC-5 fibroblast cells. The cells were pre-treated for 1 h with the indicated concentrations of SFN and for 24 h with TGF-β1 (1.0 or 5.0 ng/ml). Western blotting for phosphorylated and total form of SMAD 2/3 was performed using the cell lysates of A549 epithelial cells (a) and MRC-5 cells (b). The data are expressed as means ± standard deviation of at least three different experiments

Fig. 4

The effect of SFN on bleomycin (BLM)-induced pulmonary fibrosis. The histological results of haematoxylin and eosin, Masson’s trichrome (M-T) staining, immunohistochemistry staining of E-cad and α-SMA in lung sections (a). BLM induced extensive pulmonary inflammation and fibrosis. SFN treatment attenuated BLM-induced pulmonary fibrosis. M-T scores of lung histology (b). The M-T score increased significantly in the BLM group, and SFN treatment decreased the M-T score compared with the BLM group. The hydroxyproline assay of lung tissues (c). Collagen contents were evaluated by the hydroxyproline assay. The results were similar to the M-T scores. Image analysis of immunohistochemistry for E-cad (d) and α-SMA (e). SFN treatment restored E-cad expression and decreased the expression of α-SMA. The data are expressed as means ± standard deviation, n = 4 in each group. ^*p < 0.05, ^***p < 0.001 versus the control; ^††p < 0.01, ^†††p < 0.001 versus the BLM group

Fig. 5

The effect of SFN on TGF-β, fibronectin expression in BLM-induced pulmonary fibrosis. The protein levels of fibronectin (a) and TGF-β (b) were measured by western blot analyses. Each experiment was performed in triplicate and repeated three times. The data are expressed as means ± standard deviation, n = 4 in each group. ^**p < 0.01, ^***p < 0.001 versus the control; ^††p < 0.01, ^†††p < 0.001 versus the BLM group