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Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway
Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway Rundi Gao, Ruilin Chen, Yu Cao, Yuan Wang, Kang Song, Ya Zhang, Junchao Yang PII: DOI: Reference:
S0041-008X(16)30381-7 doi:10.1016/j.taap.2016.12.009 YTAAP 13827
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
19 July 2016 17 November 2016 13 December 2016
Please cite this article as: Gao, Rundi, Chen, Ruilin, Cao, Yu, Wang, Yuan, Song, Kang, Zhang, Ya, Yang, Junchao, Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway, Toxicology and Applied Pharmacology (2016), doi:10.1016/j.taap.2016.12.009
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ACCEPTED MANUSCRIPT Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in
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alveolar epithelial cells through Notch signaling pathway
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Rundi Gao1, Ruilin Chen1, Yu Cao1, Yuan Wang2, Kang Song1, Ya Zhang3, Junchao Yang1*
1. Department of Respiration, The First Affiliated Hospital of Zhejiang Chinese
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Zhejiang Province, China, 310006
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Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou,
2. Department of Pulmonary Function, The First Affiliated Hospital of Zhejiang
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Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District,
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Hangzhou, Zhejiang Province, China, 310006
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3. Zhejiang Chinese Medicine University, No. 548, Binwen Road, Binjiang District,
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Hangzhou, Zhejiang Province, China, 310006
*Corresponding author: Junchao Yang. Telephone: +86 571 86620305; Fax: +86 571 87077785. E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Pulmonary fibrosis is characterized by the destruction of lung tissue architecture
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and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a
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component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays
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an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of
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alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type Ⅱ epithelial cells
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RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I,
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α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin
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could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the
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inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis.
Key words: Emodin, epithelial-mesenchymal transition, Notch-1, RLE-6TN cells, pulmonary fibrosis.
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ACCEPTED MANUSCRIPT 1. Introduction Pulmonary fibrosis (PF), a consequence of various lung diseases, is characterized
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by the damage of lung tissues and an enhancement in collagen-rich extracellular
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matrix (ECM). The accumulation of ECM, damage of alveolar walls and loss of functional capillary units can result in respiratory failure (Giri, 2003). A subsequent hypothesis suggested that epithelial injury and impaired wound repair, without
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preceding inflammation, was the pathogenesis of fibrosis (Strieter and Mehrad, 2009).
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Recent evidence suggest that fibrosis in lung tissue may be derived transition of type II alveolar cells to fibroblasts. Prevention of this trans differentiation may be a key
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factor in the preservation of normal lung epithelial structure (Kim et al., 2006; Willis
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et al., 2005). Although several articles have reported the probable molecular
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mechanism of pulmonary fibrosis, current treatment for pulmonary fibrosis show poor efficacy and do not prevent or reverse the disease progression (Raghu et al., 2011).
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Emodin, a component of Chinese herbs, including Rhubarb and Polygonum cuspidatum (Dasgupta and Mukherjee, 2016; Kuo et al., 2015), has been widely used in Chinese traditional medicine. Emodin has been thought to be involved in a number of biological processes, such as antitumour (Huang et al., 2005; Iwanowycz et al., 2016; Muto et al., 2007), hepatoprotection and anti-inflammatory activities (Dong et al., 2016; Zhu et al., 2016). Recently, several reports suggest that Emodin inhibits ventricular fibrosis, pancreatic fibrosis and hepatic fibrosis (Chen et al., 2014; Wang et al., 2007; Zhan et al., 2001). Emodin also shows protective function in bleomycin induced pulmonary fibrosis (Chen et al., 2009). However, the protective mechanism
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ACCEPTED MANUSCRIPT of Emodin in pulmonary fibrosis needed be further investigated.. Alveolar epithelial cells are important target cells that can directly promote lung
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fibrosis by acquiring a mesenchymal phenotype through epithelial-mesenchymal
epithelial
cell-associated
genes
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transition (EMT). EMT is genetically characterized by a decreased expression of (E-cadherin)
and
increased
expression
of
mesenchymal cell and fibrotic-associated genes, such as Vimentin, α-smooth muscle
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actin (α-SMA), and also Collagen I (Kalluri and Neilson, 2003; Willis and Borok,
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2007; Zhu et al., 2015). Emerging evidence has demonstrated that EMT plays important role in pulmonary fibrosis (Hisatomi et al., 2012).
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Notch, a transmembrane receptor, is activated when it interacts with ligands of a
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neighbor cell, leading to proteolytic cleavage and release of Notch intracellular
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domain (NICD). NICD then enters the nucleus and regulates the expression of many Notch target genes, including Jagged (Guruharsha et al., 2012). Notch signaling
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pathway has been reported to participate in tissue fibrosis (such as pulmonary, renal and liver fibrosis) (Hu and Phan, 2016) and play a key role in modulating EMT in several malignancies (Zhou et al., 2016). However, the relationship between Notch signal pathway and Emodin in pulmonary fibrosis is yet to be investigated. In the present study, we hypothesized that Emodin may be involved in lung fibrosis by suppressing EMT. We found that treatment of alveolar type Ⅱ epithelial cells RLE-6TN with Emodin attenuated the TGF-β1-induced fibrosis and EMT-related changes. Moreover, Emodin inhibits the EMT and exerts its anti-fibrotic effects via suppression of Notch signal pathway.
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ACCEPTED MANUSCRIPT 2. Materials and Methods
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2.1 Cell line and cell culture
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Rat alveolar type Ⅱ epithelial cell line RLE-6TN (ATCC, USA) was cultured in
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DMEM medium (GIBCO, USA) supplemented with 10% fetal calf serum (GIBCO, USA), 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified incubator
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with 5% CO2 at 37 °C. For Emodin and TGF-β1 treatment, Emodin (Sigma, USA) and TGF-β1 (R&D Systerm, USA) were firstly dissolved in DMSO (Sigma, USA)
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and further diluted to 10 µg/ml or 5 ng/ml respectively, the final DMSO concentration
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was less than 0.1% (v/v).
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2.2 RNA interference
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Specific small interfering RNA (siRNA) of Notch-1 targeting NICD (activated form of Notch-1) (sense: 5’- AGG CAA CAG UGA AGA AGA ATT -3’; antisense:
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5’-UUC UUC UUC ACU GUU GCC UTT -3’) and negative control (sense: 5’-UUC UCC GAA CGU GUC ACG UTT -3’; antisense: 5’-ACG UGA CAC GUU CGG AGA ATT -3’) were purchased from GenePharma, China. Cells (1.5×105) grown on six-well plates were transfected with 100 pmol siRNA of Notch-1 (si-Notch-1) or negative control (si-NC) using 8 µl siRNA-Mate transfection reagent (GenePharma, Co., Ltd., Shanghai, China). The cells were harvested after 72 h. Western blot analyses were performed. 2.3 Cell proliferation assays After Emodin or TGF-β1 treatment, cell proliferation was measured by 5
ACCEPTED MANUSCRIPT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, RLE-6TN cells were plated in 96-well plate at 2×104 per well and cultured for 24 h,
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then the cells were treated with different concentration of Emodin (0, 2.5, 5, 10,
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20µg/ml) or 10µg/ml Emodin co-treated with 5 ng/ml TGF-β1 for 48 h. The MTT (Sigma, USA) was added to a final concentration of 0.5 mg/ml, and the cells were incubated for 4 h at 37 °C. The absorbance at 570 nm was measured by micro plate
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reader (Bio-rad, USA). Each experiment was repeated at least three times in triplicate.
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2.4 Western blot analysis
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After different treatments, cells were harvested and homogenized with lysis
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buffer (Beyotime, China). The protein concentrations were measured using BCA protein assay kit (Beyotime, China). Proteins (30 to 50 μg) were separated by 8~10%
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SDS-PAGE and transferred to nitrocellulose membrane (Millipore, USA). After blocking the non-specific antibody binding sites of membranes with 5% non-fat dry milk, the membranes were incubated with primary antibodies specific to Collagen I, α-SMA , E-cadherin, Vimentin, Notch-1, Jagged-1 and β-actin (1:1000, Cell signaling technology, USA). The membranes were further incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz, USA). The immunoblots were quantified by Image J software. 2.5 Immunofluorescence staining For the detection of Notch-1 nuclear translocation, cells (1×105 per well) were 6
ACCEPTED MANUSCRIPT seeded in 6-well glass-bottomed plate. After the cells were treated, they were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for
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15 min. Non-specific binding sites were blocked with 1% BSA in PBS for 2h. Then,
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the cells were treated with primary antibody specific to Notch-1(1:200, diluted in 1% BSA) overnight at 4 °C. Thereafter, the cells were incubated with TRITC-conjugated secondary antibody (Beyotime, China) for 1h in the dark. DAPI (Beyotime, China)
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was used to stain nuclei before capturing images. The images were acquired using a
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fluorescence microscope (Nikon, Japan). The red fluorescence indicated Notch-1 expression, and the blue fluorescence indicated nuclei.
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2.6 Statistical analysis
Data are presented as the mean ± SEM. The Student’s t test was used for
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comparison between two groups. p <0.05 and p < 0.01 were considered to indicate statistical significance. Each test data from independent experiments were repeated at
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least three times.
3. Results 3.1 Emodin inhibits cell proliferation and fibrotic markers expression in RLE-6TN cells with TGF-β stimulation To investigate the effect of Emodin in rat alveolar epithelial cell line RLE-6TN, the effects of different concentrations of Emodin on RLE-6TN cells proliferation were determined using MTT assay. The results showed that the proliferation of RLE-6TN
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ACCEPTED MANUSCRIPT cells was not significantly altered when the concentrations of Emodin were 0, 2.5 and 5 μg/ml; the lowest effective concentration was 10 μg/ml (Figure 1A). Then we
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treated RLE-6TN cells with or without 5 ng/ml TGF-β1 in the presence or absence of
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Emodin and assessed the proliferation of RLE-6TN cells. The results showed that the proliferation of RLE-6TN cells was promoted by TGF-β1 treatment in the absence of Emodin; whereas reduced by Emodin at a concentration of 10 μg/ml (Figure 1B).
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Next, we assessed the effect of Emodin on the expression of fibrosis associated
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markers, Collagen I and α-SMA. Western blot assay showed that Emodin inhibited the expression levels of Collagen I and α-SMA protein; whereas TGF-β1-induced the
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expression levels of Collagen I and α-SMA protein. Emodin could partly restore the
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promotive effect of TGF-β1 on both two markers (Figure 1C).
3.2 Emodin inhibits TGF-β1-induced the EMT in RLE-6TN cells
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Then we observed the cell phenotypes in response to Emodin or TGF-β1. Cells maintained normal phenotype in response to Emodin, compared to the control group; however, the phenotype altered in response to TGF-β1 treatment, more like fibrosis phenotype (Figure 2A). When co-treated with Emodin and TGF-β1, cell phenotype was partially restored to normal (Figure 2A). To confirm the effect of Emodin in alveolar epithelial cells, the protein levels of E-cadherin and Vimentin were evaluated using Western blot. We found that Emodin could enhance E-cadherin expression whereas decrease Vimentin expression; TGF-β1 induced Vimentin expression whereas decrease E-cadherin expression. The effect of TGF-β1 could be partly restored by the
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ACCEPTED MANUSCRIPT presence of Emodin (Figure 2B).
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3.3 Emodin inhibits TGF-β1-induced EMT by inactivating the Notch signaling
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pathway
To further investigate the role of Emodin in fibrosis and EMT, the expression pattern of Notch-associated factors was determined using Western blot. Emodin
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inhibited the expression levels of Notch-1 and Jagged-1, while TGF-β1 induced the
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expression levels of Notch-1 and Jagged-1; Emodin could partly restore the promotive effect of TGF-β1 on Notch-1 and Jagged-1 expression, suggesting that Emodin
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regulates Notch signaling pathway (Figure 3A). Next, DAPT (γ-secretase inhibitor), a
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Notch signaling pathway inhibitor, treatment was generated and then the expression
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levels of Notch-1 and Jagged-1 were evaluated. Results showed that DAPT inhibited both the expression levels of Notch-1 and Jagged-1; both DAPT and Emodin could
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partly restore the promotive effect of TGF-β1 on Notch-1 and Jagged-1 expression (Figure 3B). The expression patterns of EMT and fibrotic markers were also determined. Western blot results showed that Collagen I, α-SMA and Vimentin expression were significantly reduced, while E-cadherin expression was promoted by DAPT treatment; TGF-β1 induced Collagen I, α-SMA and Vimentin expression, while inhibited E-cadherin; when TGF-β1, DAPT and Emodin added at the same time, the effect of TGF-β1 on Collagen I, α-SMA, Vimentin and E-cadherin could almost abolished by DAPT and Emodin (Figure 3C).
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ACCEPTED MANUSCRIPT 3.4 Emodin inhibits TGF-β1-induced Notch-1 nuclear translocation in RLE-6TN cells investigate
the
effect
of
Emodin
on
inhibiting
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further
EMT,
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immunofluorescence labeling as well as Western blot was performed to determine its role in Notch-1 nuclear translocation. TGF-β1 upregulated Notch-1 expression both in nucleus and cytoplasm, Moreover, the upregulation was more obvious in nucleus,
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indicating TGF-β1 induced Notch-1 nuclear translocation. Emodin could partly
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restore TGF-β1’s promotive effect both in nucleus and cytoplasm. The restorative effect of Emodin on TGF-β1’s promotive effect was more obvious in the nucleus
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(Figure 4A). As exhibited by Western blot assay, the expression trend is consistent
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with immunofluorescence results. Emodin reduced TGF-β1-induced upregulation of
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Notch-1 expression in nucleus and cytoplasm. The restorative effect of Emodin was more obvious in nucleus than in cytoplasm (0.52-fold vs 0.66-fold) (Figure 4B),
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suggesting that Emodin inhibited TGF-β1-induced Notch-1 nuclear translocation in RLE-6TN cells.
3.5 Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells To further explain the molecular mechanism by which Emodin inhibits EMT, Western blot was performed. Si-Notch-1 (targeting to Notch-1 activated form NICD) was generated to inhibit Notch-1 expression, and the inhibitory efficiency was verified using Western blot (Figure 5A). si-NC/si-Notch-1 was transfected into
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ACCEPTED MANUSCRIPT RLE-6TN cells with or without Emodin treatment. Then the expression patterns of EMT and fibrosis-associated markers were explored using Western blot. As shown in
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fig.5B, under TGF-β1 treatment, the expression levels of the mesenchymal
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phenotypic markers and fibrotic markers, Vimentin, Collagen I and α-SMA, were reduced by si-Notch-1 transfection or Emodin respectively, and even more significantly reduced in si-Notch-1 + Emodin group; while the expression of epithelial
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phenotypic marker, E-cadherin, was promoted by si-Notch-1 transfection or Emodin
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treatment respectively, and more significantly promoted in si-Notch-1 + Emodin
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4. Discussion
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group (Figure 5B).
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In this study, we found that Emodin could inhibit the EMT-related changes in rat alveolar epithelial cell line RLE-6TN cells. Some studies have reported that TGF-β1
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could induce EMT in pulmonary fibrosis; TGF-β1-induced EMT might be the major factor to the process of collagen production and fibrosis. In fact, TGF-β1 treatment on epithelial cells in culture is an efficient way to induce EMT in various epithelial cells (Kasai et al., 2005; Willis and Borok, 2007). Our results demonstrated that a treatment with TGF-β1 (5 ng/ml) could promote EMT marked by the increase of Vimentin, the decrease of E-cadherin, and the acquisition of an EMT phenotype. Fibrotic markers Collagen I and α-SMA were also upregulated under TGF-β1 treatment. Emodin is a major bioactive compound in Rhubarb and Polygonum cuspidatum. Emodin has been shown to have a number of biological activities, such as, anti-microbial,
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ACCEPTED MANUSCRIPT anti-inflammatory, anti-tumor, anti-fibrosis (Lin et al., 2015; Zhu et al., 2016). It has been reported that Emodin could alleviate bleomycin-induced pulmonary fibrosis in
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rats via TGF-β1/ADAMTS-1signaling pathway (Liu et al., 2016). Emodin also could
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inhibit lung fibroblast proliferation and induce cell cycle arrest, which may contribute to prevention of pulmonary fibrosis (Qu et al., 2004). In our study, Emodin inhibited the proliferation of alveolar epithelial cell RLE-6TN in a concentration-dependent
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manner, and 10 μg/ml Emodin co-cultured with TGF-β1 in RLE-6TN cells attenuated
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the proliferation which could be promoted by TGF-β1. Moreover, Emodin also attenuated the expression levels of Collagen I, α-SMA and Vimentin, increased the
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TGF-β1-induced EMT.
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expression of E-cadherin. These data suggested that Emodin could inhibit the
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The TGF-β1-induced EMT might be regulated through different pathways, including Smads, mitogen-activated protein kinase (MAPK), and Notch signaling
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pathways (Li et al., 2013; Wei et al., 2013; Yang et al., 2015). Wang et al. described the role of Notch signaling pathway in EMT, and cataloged how its deregulation is involved in EMT and tumor aggressiveness (Wang et al., 2010). To further investigate the mechanism by which Emodin inhibits EMT, the exact role of Emodin in regulating TGF-β1-induced EMT was investigated. Our results verified that TGF-β1 promoted the expression levels of Notch-1, one of the Notch receptors, and one of its ligands, Jagged-1. The EMT inhibitory effect of Emodin has been reported in several carcinoma cells (Hu et al., 2016; Way et al., 2014). Similarly, in our study, Emodin also inhibited TGF-β1-induced EMT by
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ACCEPTED MANUSCRIPT decreased the expression levels of Notch-1 and Jagged-1. Moreover, the Notch-1 specific inhibitor DAPT significantly strengthened the effect of Emodin on Notch
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signaling pathway in TGF-β1-induced EMT. These results suggest that Emodin
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partially suppressed the TGF-β1-induced EMT in RLE-6TN cells through the TGF-β1/Notch signaling pathway. TGF-β1 induced Notch-1 nuclear translocation, while this upregulation of Notch-1 by TGF-β1 could be partly restored by Emodin
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both in nucleus and cytoplasm. The restorative effect of Emodin on TGF-β1’s
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promotive effect was more obvious in nucleus, indicating that Emodin blocked TGF-β1-induced Notch-1 nucleus translocation which is a key step of activation of
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Notch-1, consistent with previous studies (Licciardello et al., 2015). Knocking down
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the expression of Notch-1 by si-Notch-1, which specially targeting Notch-1 activated
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form NICD could partially strengthen the effect of Emodin on inhibiting the TGF-β1-induced EMT. Several reports demonstrated the regulatory effect of Emodin
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on Notch pathway (Deng et al., 2015; Kim et al., 2015), which is consistent with our findings in the present study. However, the data in the present study indicated that Notch pathway plays a role in the process of Emodin regulating alveolar epithelial cells EMT. Emodin could inhibit the alveolar epithelial cells EMT partially through inhibiting Notch-1 nucleus translocation and activation. In conclusion, we found the inhibitory effect of Emodin on TGF-β1-induced EMT, and demonstrated the mechanism by which Emodin exerts its role in regulating EMT. The present study provided evidence of the potential therapeutic use of Emodin in the prevention and or control of pulmonary fibrosis.
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Acknowledgements
and
Natural
Science
Foundation
of
Zhejiang
Province
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(No.81302937)
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This work was supported by National Natural Science Foundation of China
(No.LY13H270009).
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A.G., Richeldi, L., Selman, M., Dudden, R.F., Griss, B.S., Protzko, S.L., Schunemann, H.J., 2011. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med 183, 788-824. Strieter, R.M., Mehrad, B., 2009. New mechanisms of pulmonary fibrosis. Chest 136, 1364-1370. Wang, C.H., Gao, Z.Q., Ye, B., Cai, J.T., Xie, C.G., Qian, K.D., Du, Q., 2007. Effect of emodin on pancreatic fibrosis in rats. World J Gastroenterol 13, 378-382. Wang, Z., Li, Y., Kong, D., Sarkar, F.H., 2010. The role of Notch signaling pathway in epithelial-mesenchymal transition (EMT) during development and tumor aggressiveness. Curr Drug Targets 11, 745-751. Way, T.-D., Huang, J.-T., Chou, C.-H., Huang, C.-H., Yang, M.-H., Ho, C.-T., 2014. Emodin represses TWIST1-induced epithelial–mesenchymal transitions in head and neck squamous cell carcinoma cells by inhibiting the β-catenin and Akt pathways. European journal of cancer 50, 366-378. Wei, J., Li, Z., Chen, W., Ma, C., Zhan, F., Wu, W., Peng, Y., 2013. AEG-1 participates in TGF-beta1-induced EMT through p38 MAPK activation. Cell Biol Int 37, 1016-1021. Willis, B.C., Borok, Z., 2007. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 293, L525-534. Willis, B.C., Liebler, J.M., Luby-Phelps, K., Nicholson, A.G., Crandall, E.D., du Bois, R.M., Borok, Z., 2005. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 166, 1321-1332. Yang, H., Zhan, L., Yang, T., Wang, L., Li, C., Zhao, J., Lei, Z., Li, X., Zhang, H.T., 2015. Ski prevents TGF-beta-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer. Oncol Rep 34, 87-94. Zhan, Y., Wei, H., Wang, Z., Huang, X., Xu, Q., Li, D., Lu, H., 2001. Effects of emodin on hepatic fibrosis in rats. Zhonghua Gan Zang Bing Za Zhi 9, 235-236. Zhou, J., Jain, S., Azad, A.K., Xu, X., Yu, H.C., Xu, Z., Godbout, R., Fu, Y., 2016. Notch and TGFβ form a positive regulatory loop and regulate EMT in epithelial ovarian cancer cells. Cellular signalling 28, 838-849. Zhu, T., Zhang, W., Feng, S.-j., Yu, H.-p., 2016. Emodin suppresses LPS-induced inflammation in RAW264. 7 cells through a PPARγ-dependent pathway. International immunopharmacology 34, 16-24. Zhu, X., Zhong, J., Zhao, Z., Sheng, J., Wang, J., Liu, J., Cui, K., Chang, J., Zhao, H., Wong, S., 2015. Epithelial derived CTGF promotes breast tumor progression via inducing EMT and collagen I fibers deposition. Oncotarget 6, 25320-25338.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Emodin inhibits cell proliferation and fibrotic markers expression in
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RLE-6TN cells with TGF-β stimulation. RLE-6TN cells were treated with different
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concentration of Emodin (0, 2.5, 5, 10, 20 μg/ml) or co-treated with TGF-β1 (5 ng/ml) for 48h. (A) and (B) Cell proliferation was measured by MTT assay. Proliferation of control cells was set as 100%. (C) Protein expression of Collagen I and α-SMA was
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determined by western blot. β-actin was used as the protein loading control. The
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densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments. Data are mean ± SEM. * P <0.05 and
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** p<0.01 vs. control group. ## P <0.01 vs. TGF-β1 group.
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Figure 2 Effect of Emodin on morphological transformation and EMT markers in RLE-6TN cells with TGF-β1 stimulation. Cells were treated with 10μg/ml
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Emodin or co-treated with 5 ng/ml TGF-β1 for 48 h. (A) Phase contrast images of RLE-6TN cells. (B) Protein expression of E-cadherin and Vimentin was determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments.. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. # P <0.05 and ## P <0.01 vs. TGF-β1 group.
Figure 3 Emodin inhibits TGF-β1-induced EMT by inactivating the Notch signaling pathway. RLE-6TN cells were treated with 10 μg/ml Emodin or co-treated
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ACCEPTED MANUSCRIPT with 5 ng/ml TGF-β1 for 48h. (A) Protein expression of Notch-1 and Jagged-1 was determined by western blot. (B) and (C) after exposure to a special Notch signal
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pathway inhibitor, DAPT (1 μg/ml). Protein expression of Notch-1, Jagged-1,
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Collagen I, α-SMA, E-cadherin and Vimentin were determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate
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<0.05 and ## P <0.01 vs. TGF-β1 group.
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experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. # P
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Figure 4 The effect of Emodin on Notch nuclear translocation. RLE-6TN cells
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were treated with 5 ng/ml TGF-β1 or co-treated with 10 μg/ml Emodin for 48h. (A)
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RLE-6TN cells stained for Notch-1 (red) and nuclei (blue). Magnification: 1000×. Scale bar is 20 μm. (B) The protein expression of Notch 1 in nuclei and cytoplasm
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was determined by western blot. Histone H3 (nuclei) and β-actin (cytoplasm) were used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. ## P <0.01 vs. TGF-β1 group.
Figure 5 knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. (A) RLE-6TN cells were transfected with 100 pmol siRNA negative control (si-NC) or specific siRNA of Notch-1 (si-Notch-1)
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ACCEPTED MANUSCRIPT for 72 h, respectively, the siRNA interference effect on Notch-1 expression was measured by western blot. (B) RLE-6TN cells were transfected with siRNAs for 24 h
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then treated with 5 ng/ml TGF-β1 or co-treated with 10 μg/ml Emodin for 48h. The
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expression of Collagen I, α-SMA, E-cadherin and Vimentin were determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from
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at least 3 separate experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs.
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control group. # P <0.05 and ## P <0.01 vs. TGF-β1 group.
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Highlights Emodin inhibits TGF-β1-induced EMT in alveolar epithelial cells.
Emodin regulates the expression patterns of the Notch signaling pathway-related
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factors.
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Emodin inhibits TGF-β1-induced Notch-1 nucleus translocation and activation.
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S0041-008X(16)30381-7 doi:10.1016/j.taap.2016.12.009 YTAAP 13827
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
19 July 2016 17 November 2016 13 December 2016
Please cite this article as: Gao, Rundi, Chen, Ruilin, Cao, Yu, Wang, Yuan, Song, Kang, Zhang, Ya, Yang, Junchao, Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway, Toxicology and Applied Pharmacology (2016), doi:10.1016/j.taap.2016.12.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in
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alveolar epithelial cells through Notch signaling pathway
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Rundi Gao1, Ruilin Chen1, Yu Cao1, Yuan Wang2, Kang Song1, Ya Zhang3, Junchao Yang1*
1. Department of Respiration, The First Affiliated Hospital of Zhejiang Chinese
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Zhejiang Province, China, 310006
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Medicine University, NO. 56, Youdian Road, Shangcheng District, Hangzhou,
2. Department of Pulmonary Function, The First Affiliated Hospital of Zhejiang
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Chinese Medicine University, NO. 56, Youdian Road, Shangcheng District,
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Hangzhou, Zhejiang Province, China, 310006
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3. Zhejiang Chinese Medicine University, No. 548, Binwen Road, Binjiang District,
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Hangzhou, Zhejiang Province, China, 310006
*Corresponding author: Junchao Yang. Telephone: +86 571 86620305; Fax: +86 571 87077785. E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Pulmonary fibrosis is characterized by the destruction of lung tissue architecture
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and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a
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component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays
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an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of
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alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type Ⅱ epithelial cells
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RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I,
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α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin
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could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the
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inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis.
Key words: Emodin, epithelial-mesenchymal transition, Notch-1, RLE-6TN cells, pulmonary fibrosis.
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ACCEPTED MANUSCRIPT 1. Introduction Pulmonary fibrosis (PF), a consequence of various lung diseases, is characterized
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by the damage of lung tissues and an enhancement in collagen-rich extracellular
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matrix (ECM). The accumulation of ECM, damage of alveolar walls and loss of functional capillary units can result in respiratory failure (Giri, 2003). A subsequent hypothesis suggested that epithelial injury and impaired wound repair, without
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preceding inflammation, was the pathogenesis of fibrosis (Strieter and Mehrad, 2009).
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Recent evidence suggest that fibrosis in lung tissue may be derived transition of type II alveolar cells to fibroblasts. Prevention of this trans differentiation may be a key
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factor in the preservation of normal lung epithelial structure (Kim et al., 2006; Willis
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et al., 2005). Although several articles have reported the probable molecular
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mechanism of pulmonary fibrosis, current treatment for pulmonary fibrosis show poor efficacy and do not prevent or reverse the disease progression (Raghu et al., 2011).
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Emodin, a component of Chinese herbs, including Rhubarb and Polygonum cuspidatum (Dasgupta and Mukherjee, 2016; Kuo et al., 2015), has been widely used in Chinese traditional medicine. Emodin has been thought to be involved in a number of biological processes, such as antitumour (Huang et al., 2005; Iwanowycz et al., 2016; Muto et al., 2007), hepatoprotection and anti-inflammatory activities (Dong et al., 2016; Zhu et al., 2016). Recently, several reports suggest that Emodin inhibits ventricular fibrosis, pancreatic fibrosis and hepatic fibrosis (Chen et al., 2014; Wang et al., 2007; Zhan et al., 2001). Emodin also shows protective function in bleomycin induced pulmonary fibrosis (Chen et al., 2009). However, the protective mechanism
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ACCEPTED MANUSCRIPT of Emodin in pulmonary fibrosis needed be further investigated.. Alveolar epithelial cells are important target cells that can directly promote lung
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fibrosis by acquiring a mesenchymal phenotype through epithelial-mesenchymal
epithelial
cell-associated
genes
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transition (EMT). EMT is genetically characterized by a decreased expression of (E-cadherin)
and
increased
expression
of
mesenchymal cell and fibrotic-associated genes, such as Vimentin, α-smooth muscle
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actin (α-SMA), and also Collagen I (Kalluri and Neilson, 2003; Willis and Borok,
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2007; Zhu et al., 2015). Emerging evidence has demonstrated that EMT plays important role in pulmonary fibrosis (Hisatomi et al., 2012).
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Notch, a transmembrane receptor, is activated when it interacts with ligands of a
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neighbor cell, leading to proteolytic cleavage and release of Notch intracellular
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domain (NICD). NICD then enters the nucleus and regulates the expression of many Notch target genes, including Jagged (Guruharsha et al., 2012). Notch signaling
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pathway has been reported to participate in tissue fibrosis (such as pulmonary, renal and liver fibrosis) (Hu and Phan, 2016) and play a key role in modulating EMT in several malignancies (Zhou et al., 2016). However, the relationship between Notch signal pathway and Emodin in pulmonary fibrosis is yet to be investigated. In the present study, we hypothesized that Emodin may be involved in lung fibrosis by suppressing EMT. We found that treatment of alveolar type Ⅱ epithelial cells RLE-6TN with Emodin attenuated the TGF-β1-induced fibrosis and EMT-related changes. Moreover, Emodin inhibits the EMT and exerts its anti-fibrotic effects via suppression of Notch signal pathway.
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ACCEPTED MANUSCRIPT 2. Materials and Methods
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2.1 Cell line and cell culture
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Rat alveolar type Ⅱ epithelial cell line RLE-6TN (ATCC, USA) was cultured in
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DMEM medium (GIBCO, USA) supplemented with 10% fetal calf serum (GIBCO, USA), 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified incubator
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with 5% CO2 at 37 °C. For Emodin and TGF-β1 treatment, Emodin (Sigma, USA) and TGF-β1 (R&D Systerm, USA) were firstly dissolved in DMSO (Sigma, USA)
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and further diluted to 10 µg/ml or 5 ng/ml respectively, the final DMSO concentration
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was less than 0.1% (v/v).
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2.2 RNA interference
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Specific small interfering RNA (siRNA) of Notch-1 targeting NICD (activated form of Notch-1) (sense: 5’- AGG CAA CAG UGA AGA AGA ATT -3’; antisense:
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5’-UUC UUC UUC ACU GUU GCC UTT -3’) and negative control (sense: 5’-UUC UCC GAA CGU GUC ACG UTT -3’; antisense: 5’-ACG UGA CAC GUU CGG AGA ATT -3’) were purchased from GenePharma, China. Cells (1.5×105) grown on six-well plates were transfected with 100 pmol siRNA of Notch-1 (si-Notch-1) or negative control (si-NC) using 8 µl siRNA-Mate transfection reagent (GenePharma, Co., Ltd., Shanghai, China). The cells were harvested after 72 h. Western blot analyses were performed. 2.3 Cell proliferation assays After Emodin or TGF-β1 treatment, cell proliferation was measured by 5
ACCEPTED MANUSCRIPT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, RLE-6TN cells were plated in 96-well plate at 2×104 per well and cultured for 24 h,
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then the cells were treated with different concentration of Emodin (0, 2.5, 5, 10,
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20µg/ml) or 10µg/ml Emodin co-treated with 5 ng/ml TGF-β1 for 48 h. The MTT (Sigma, USA) was added to a final concentration of 0.5 mg/ml, and the cells were incubated for 4 h at 37 °C. The absorbance at 570 nm was measured by micro plate
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reader (Bio-rad, USA). Each experiment was repeated at least three times in triplicate.
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2.4 Western blot analysis
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After different treatments, cells were harvested and homogenized with lysis
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buffer (Beyotime, China). The protein concentrations were measured using BCA protein assay kit (Beyotime, China). Proteins (30 to 50 μg) were separated by 8~10%
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SDS-PAGE and transferred to nitrocellulose membrane (Millipore, USA). After blocking the non-specific antibody binding sites of membranes with 5% non-fat dry milk, the membranes were incubated with primary antibodies specific to Collagen I, α-SMA , E-cadherin, Vimentin, Notch-1, Jagged-1 and β-actin (1:1000, Cell signaling technology, USA). The membranes were further incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz, USA). The immunoblots were quantified by Image J software. 2.5 Immunofluorescence staining For the detection of Notch-1 nuclear translocation, cells (1×105 per well) were 6
ACCEPTED MANUSCRIPT seeded in 6-well glass-bottomed plate. After the cells were treated, they were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for
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15 min. Non-specific binding sites were blocked with 1% BSA in PBS for 2h. Then,
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the cells were treated with primary antibody specific to Notch-1(1:200, diluted in 1% BSA) overnight at 4 °C. Thereafter, the cells were incubated with TRITC-conjugated secondary antibody (Beyotime, China) for 1h in the dark. DAPI (Beyotime, China)
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was used to stain nuclei before capturing images. The images were acquired using a
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fluorescence microscope (Nikon, Japan). The red fluorescence indicated Notch-1 expression, and the blue fluorescence indicated nuclei.
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2.6 Statistical analysis
Data are presented as the mean ± SEM. The Student’s t test was used for
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comparison between two groups. p <0.05 and p < 0.01 were considered to indicate statistical significance. Each test data from independent experiments were repeated at
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least three times.
3. Results 3.1 Emodin inhibits cell proliferation and fibrotic markers expression in RLE-6TN cells with TGF-β stimulation To investigate the effect of Emodin in rat alveolar epithelial cell line RLE-6TN, the effects of different concentrations of Emodin on RLE-6TN cells proliferation were determined using MTT assay. The results showed that the proliferation of RLE-6TN
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ACCEPTED MANUSCRIPT cells was not significantly altered when the concentrations of Emodin were 0, 2.5 and 5 μg/ml; the lowest effective concentration was 10 μg/ml (Figure 1A). Then we
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treated RLE-6TN cells with or without 5 ng/ml TGF-β1 in the presence or absence of
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Emodin and assessed the proliferation of RLE-6TN cells. The results showed that the proliferation of RLE-6TN cells was promoted by TGF-β1 treatment in the absence of Emodin; whereas reduced by Emodin at a concentration of 10 μg/ml (Figure 1B).
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Next, we assessed the effect of Emodin on the expression of fibrosis associated
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markers, Collagen I and α-SMA. Western blot assay showed that Emodin inhibited the expression levels of Collagen I and α-SMA protein; whereas TGF-β1-induced the
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expression levels of Collagen I and α-SMA protein. Emodin could partly restore the
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promotive effect of TGF-β1 on both two markers (Figure 1C).
3.2 Emodin inhibits TGF-β1-induced the EMT in RLE-6TN cells
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Then we observed the cell phenotypes in response to Emodin or TGF-β1. Cells maintained normal phenotype in response to Emodin, compared to the control group; however, the phenotype altered in response to TGF-β1 treatment, more like fibrosis phenotype (Figure 2A). When co-treated with Emodin and TGF-β1, cell phenotype was partially restored to normal (Figure 2A). To confirm the effect of Emodin in alveolar epithelial cells, the protein levels of E-cadherin and Vimentin were evaluated using Western blot. We found that Emodin could enhance E-cadherin expression whereas decrease Vimentin expression; TGF-β1 induced Vimentin expression whereas decrease E-cadherin expression. The effect of TGF-β1 could be partly restored by the
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3.3 Emodin inhibits TGF-β1-induced EMT by inactivating the Notch signaling
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pathway
To further investigate the role of Emodin in fibrosis and EMT, the expression pattern of Notch-associated factors was determined using Western blot. Emodin
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inhibited the expression levels of Notch-1 and Jagged-1, while TGF-β1 induced the
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expression levels of Notch-1 and Jagged-1; Emodin could partly restore the promotive effect of TGF-β1 on Notch-1 and Jagged-1 expression, suggesting that Emodin
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regulates Notch signaling pathway (Figure 3A). Next, DAPT (γ-secretase inhibitor), a
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Notch signaling pathway inhibitor, treatment was generated and then the expression
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levels of Notch-1 and Jagged-1 were evaluated. Results showed that DAPT inhibited both the expression levels of Notch-1 and Jagged-1; both DAPT and Emodin could
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partly restore the promotive effect of TGF-β1 on Notch-1 and Jagged-1 expression (Figure 3B). The expression patterns of EMT and fibrotic markers were also determined. Western blot results showed that Collagen I, α-SMA and Vimentin expression were significantly reduced, while E-cadherin expression was promoted by DAPT treatment; TGF-β1 induced Collagen I, α-SMA and Vimentin expression, while inhibited E-cadherin; when TGF-β1, DAPT and Emodin added at the same time, the effect of TGF-β1 on Collagen I, α-SMA, Vimentin and E-cadherin could almost abolished by DAPT and Emodin (Figure 3C).
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ACCEPTED MANUSCRIPT 3.4 Emodin inhibits TGF-β1-induced Notch-1 nuclear translocation in RLE-6TN cells investigate
the
effect
of
Emodin
on
inhibiting
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further
EMT,
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immunofluorescence labeling as well as Western blot was performed to determine its role in Notch-1 nuclear translocation. TGF-β1 upregulated Notch-1 expression both in nucleus and cytoplasm, Moreover, the upregulation was more obvious in nucleus,
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indicating TGF-β1 induced Notch-1 nuclear translocation. Emodin could partly
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restore TGF-β1’s promotive effect both in nucleus and cytoplasm. The restorative effect of Emodin on TGF-β1’s promotive effect was more obvious in the nucleus
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(Figure 4A). As exhibited by Western blot assay, the expression trend is consistent
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with immunofluorescence results. Emodin reduced TGF-β1-induced upregulation of
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Notch-1 expression in nucleus and cytoplasm. The restorative effect of Emodin was more obvious in nucleus than in cytoplasm (0.52-fold vs 0.66-fold) (Figure 4B),
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suggesting that Emodin inhibited TGF-β1-induced Notch-1 nuclear translocation in RLE-6TN cells.
3.5 Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells To further explain the molecular mechanism by which Emodin inhibits EMT, Western blot was performed. Si-Notch-1 (targeting to Notch-1 activated form NICD) was generated to inhibit Notch-1 expression, and the inhibitory efficiency was verified using Western blot (Figure 5A). si-NC/si-Notch-1 was transfected into
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ACCEPTED MANUSCRIPT RLE-6TN cells with or without Emodin treatment. Then the expression patterns of EMT and fibrosis-associated markers were explored using Western blot. As shown in
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phenotypic markers and fibrotic markers, Vimentin, Collagen I and α-SMA, were reduced by si-Notch-1 transfection or Emodin respectively, and even more significantly reduced in si-Notch-1 + Emodin group; while the expression of epithelial
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phenotypic marker, E-cadherin, was promoted by si-Notch-1 transfection or Emodin
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treatment respectively, and more significantly promoted in si-Notch-1 + Emodin
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4. Discussion
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group (Figure 5B).
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In this study, we found that Emodin could inhibit the EMT-related changes in rat alveolar epithelial cell line RLE-6TN cells. Some studies have reported that TGF-β1
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could induce EMT in pulmonary fibrosis; TGF-β1-induced EMT might be the major factor to the process of collagen production and fibrosis. In fact, TGF-β1 treatment on epithelial cells in culture is an efficient way to induce EMT in various epithelial cells (Kasai et al., 2005; Willis and Borok, 2007). Our results demonstrated that a treatment with TGF-β1 (5 ng/ml) could promote EMT marked by the increase of Vimentin, the decrease of E-cadherin, and the acquisition of an EMT phenotype. Fibrotic markers Collagen I and α-SMA were also upregulated under TGF-β1 treatment. Emodin is a major bioactive compound in Rhubarb and Polygonum cuspidatum. Emodin has been shown to have a number of biological activities, such as, anti-microbial,
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ACCEPTED MANUSCRIPT anti-inflammatory, anti-tumor, anti-fibrosis (Lin et al., 2015; Zhu et al., 2016). It has been reported that Emodin could alleviate bleomycin-induced pulmonary fibrosis in
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rats via TGF-β1/ADAMTS-1signaling pathway (Liu et al., 2016). Emodin also could
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inhibit lung fibroblast proliferation and induce cell cycle arrest, which may contribute to prevention of pulmonary fibrosis (Qu et al., 2004). In our study, Emodin inhibited the proliferation of alveolar epithelial cell RLE-6TN in a concentration-dependent
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manner, and 10 μg/ml Emodin co-cultured with TGF-β1 in RLE-6TN cells attenuated
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the proliferation which could be promoted by TGF-β1. Moreover, Emodin also attenuated the expression levels of Collagen I, α-SMA and Vimentin, increased the
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TGF-β1-induced EMT.
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expression of E-cadherin. These data suggested that Emodin could inhibit the
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The TGF-β1-induced EMT might be regulated through different pathways, including Smads, mitogen-activated protein kinase (MAPK), and Notch signaling
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pathways (Li et al., 2013; Wei et al., 2013; Yang et al., 2015). Wang et al. described the role of Notch signaling pathway in EMT, and cataloged how its deregulation is involved in EMT and tumor aggressiveness (Wang et al., 2010). To further investigate the mechanism by which Emodin inhibits EMT, the exact role of Emodin in regulating TGF-β1-induced EMT was investigated. Our results verified that TGF-β1 promoted the expression levels of Notch-1, one of the Notch receptors, and one of its ligands, Jagged-1. The EMT inhibitory effect of Emodin has been reported in several carcinoma cells (Hu et al., 2016; Way et al., 2014). Similarly, in our study, Emodin also inhibited TGF-β1-induced EMT by
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signaling pathway in TGF-β1-induced EMT. These results suggest that Emodin
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partially suppressed the TGF-β1-induced EMT in RLE-6TN cells through the TGF-β1/Notch signaling pathway. TGF-β1 induced Notch-1 nuclear translocation, while this upregulation of Notch-1 by TGF-β1 could be partly restored by Emodin
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both in nucleus and cytoplasm. The restorative effect of Emodin on TGF-β1’s
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promotive effect was more obvious in nucleus, indicating that Emodin blocked TGF-β1-induced Notch-1 nucleus translocation which is a key step of activation of
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Notch-1, consistent with previous studies (Licciardello et al., 2015). Knocking down
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the expression of Notch-1 by si-Notch-1, which specially targeting Notch-1 activated
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form NICD could partially strengthen the effect of Emodin on inhibiting the TGF-β1-induced EMT. Several reports demonstrated the regulatory effect of Emodin
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on Notch pathway (Deng et al., 2015; Kim et al., 2015), which is consistent with our findings in the present study. However, the data in the present study indicated that Notch pathway plays a role in the process of Emodin regulating alveolar epithelial cells EMT. Emodin could inhibit the alveolar epithelial cells EMT partially through inhibiting Notch-1 nucleus translocation and activation. In conclusion, we found the inhibitory effect of Emodin on TGF-β1-induced EMT, and demonstrated the mechanism by which Emodin exerts its role in regulating EMT. The present study provided evidence of the potential therapeutic use of Emodin in the prevention and or control of pulmonary fibrosis.
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Acknowledgements
and
Natural
Science
Foundation
of
Zhejiang
Province
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(No.81302937)
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This work was supported by National Natural Science Foundation of China
(No.LY13H270009).
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References
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Emodin inhibits cell proliferation and fibrotic markers expression in
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RLE-6TN cells with TGF-β stimulation. RLE-6TN cells were treated with different
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concentration of Emodin (0, 2.5, 5, 10, 20 μg/ml) or co-treated with TGF-β1 (5 ng/ml) for 48h. (A) and (B) Cell proliferation was measured by MTT assay. Proliferation of control cells was set as 100%. (C) Protein expression of Collagen I and α-SMA was
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determined by western blot. β-actin was used as the protein loading control. The
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densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments. Data are mean ± SEM. * P <0.05 and
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** p<0.01 vs. control group. ## P <0.01 vs. TGF-β1 group.
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Figure 2 Effect of Emodin on morphological transformation and EMT markers in RLE-6TN cells with TGF-β1 stimulation. Cells were treated with 10μg/ml
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Emodin or co-treated with 5 ng/ml TGF-β1 for 48 h. (A) Phase contrast images of RLE-6TN cells. (B) Protein expression of E-cadherin and Vimentin was determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments.. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. # P <0.05 and ## P <0.01 vs. TGF-β1 group.
Figure 3 Emodin inhibits TGF-β1-induced EMT by inactivating the Notch signaling pathway. RLE-6TN cells were treated with 10 μg/ml Emodin or co-treated
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pathway inhibitor, DAPT (1 μg/ml). Protein expression of Notch-1, Jagged-1,
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Collagen I, α-SMA, E-cadherin and Vimentin were determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate
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<0.05 and ## P <0.01 vs. TGF-β1 group.
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experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. # P
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Figure 4 The effect of Emodin on Notch nuclear translocation. RLE-6TN cells
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were treated with 5 ng/ml TGF-β1 or co-treated with 10 μg/ml Emodin for 48h. (A)
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RLE-6TN cells stained for Notch-1 (red) and nuclei (blue). Magnification: 1000×. Scale bar is 20 μm. (B) The protein expression of Notch 1 in nuclei and cytoplasm
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was determined by western blot. Histone H3 (nuclei) and β-actin (cytoplasm) were used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from at least 3 separate experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs. control group. ## P <0.01 vs. TGF-β1 group.
Figure 5 knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. (A) RLE-6TN cells were transfected with 100 pmol siRNA negative control (si-NC) or specific siRNA of Notch-1 (si-Notch-1)
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ACCEPTED MANUSCRIPT for 72 h, respectively, the siRNA interference effect on Notch-1 expression was measured by western blot. (B) RLE-6TN cells were transfected with siRNAs for 24 h
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then treated with 5 ng/ml TGF-β1 or co-treated with 10 μg/ml Emodin for 48h. The
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expression of Collagen I, α-SMA, E-cadherin and Vimentin were determined by western blot. β-actin was used as the protein loading control. The densitometric analysis results are shown in the right panels. All reported results were reported from
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at least 3 separate experiments. Data are mean± SEM. * P <0.05 and ** P <0.01 vs.
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control group. # P <0.05 and ## P <0.01 vs. TGF-β1 group.
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Highlights Emodin inhibits TGF-β1-induced EMT in alveolar epithelial cells.
Emodin regulates the expression patterns of the Notch signaling pathway-related
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factors.
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Emodin inhibits TGF-β1-induced Notch-1 nucleus translocation and activation.
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