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. 2021 Oct 15;22(20):11152.
doi: 10.3390/ijms222011152.

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Kai-Wei Chang  1   2 , Xiang Zhang  3 , Shih-Chao Lin  4 , Yu-Chao Lin  5   6   7 , Chia-Hsiang Li  5   6   7 , Ivan Akhrymuk  8 , Sheng-Hao Lin  9   10   11 , Chi-Chien Lin  2   12   13   14   15
Affiliations

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Kai-Wei Chang et al. Int J Mol Sci. .

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic _target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.

Keywords: MAPK; Smad2/3; atractylodin; epithelial-mesenchymal transition; idiopathic pulmonary fibrosis; transforming growth factor-beta 1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Atractylodin caused neglectable cytotoxicity on A549 cells with or without TGF-β1. (A) The molecular structure of atractylodin. (B) The cell viability of A549 cells treated with ATL (0, 25, 50, 100 μM) for 24 h was determined by MTT assay. (C) The cell viability of A549 cells treated with different concentrations of ATL in the presence of TGFβ1 (2 ng/mL) was measured by MTT assay. Values represent the mean ± SEM from triplicate samples for each treatment. (D) 3D structure of the docking model. Atractylodin is indicated in blue; the homodimer is composed of TGF-β1A (in tan) and TGF-β1B (in grey); amino acid residuals interacting with TGF-β1 receptor are labeled in pink for Trp30, Trp32, Tyr90 of TGF1-β1A and in orange for Lys60 of TGF-β1B. Values represent the mean ± SEM from triplicate samples for each treatment.
Figure 2
Figure 2
Effect of atractylodin stymied on TGF-β1-induced EMT-associated protein expressions in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 24 h. Cells treated with DMSO were set up as the control groups. (A) Protein expression levels of N-cadherin, E-cadherin, α-SMA, and vimentin were measured by Western blot assay. (B) Quantitation of Western blot signal intensities by ImageJ software. (C) The transcriptional expressions of type I collagen and (D) type III collagen were conducted by RT-qPCR. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).
Figure 3
Figure 3
Atractylodin moderately reduced EMT-related transcription factor expression in TGF-β1-treated A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 24 h. Cells treated with DMSO were set up as the control group. Real-time PCR was exploited for quantifying the expressional changes of EMT-related transcription factors, including Snail, Slug, Twist, ZEB1, and ZEB2. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).
Figure 4
Figure 4
Atractylodin suppressed Smad-dependent pathway activation triggered by TGF-β1 in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 6 h. Cells treated with DMSO were set up as the control group. (A) Protein expression levels of p-Smad2, p-Smad3, Smad2, and Smad3 were measured by Western blot assay. (B) Quantitation of Western blot signal intensities with ImageJ software. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).
Figure 5
Figure 5
Atractylodin reduced Smad-independent pathway activated by TGF-β1 in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 6 h. Cells treated with DMSO were set up as the control group. (A) Protein expression levels of phospho- and non-phospho- p38, JNK, ERK, and AKT were measured by Western blot assay. (B) Quantitation of Western blot signal intensities by ImageJ software. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).
Figure 6
Figure 6
Atractylodin ameliorated BLM-induced pulmonary fibrosis. (A) The body weight changes in BLM-treated mice received ATL treatment 0, 50, and 100 mg/kg. (B) The lung function test for Penh value was performed by plethysmograph on day 21. (C) Numbers of total inflammatory cells and (D) immune cells of neutrophils, lymphocytes as well as mononuclear cells in BALF were stained with Wright-Giemsa stain and counted under the microscopy. Data are expressed as mean ± SEM of five mice in each group. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 versus vehicle-treated BLM model group (as control group), as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method). (E) Pulmonary pathological changes in tissues from BLM treated mice with or without ATL treatments were examined followed by H&E staining (original magnifications, 400×). (F) Collagen deposition in lung tissues was visualized and examined by Masson’s trichrome staining (original magnifications, 400×).
Figure 7
Figure 7
Atractylodin reduced BLM-induced EMT in mice pulmonary tissues. Lung tissue homogenates were collected on day 21 from each group of mice. (A) Relative mRNA expression levels of E-cadherin, α-SMA, and vimentin were measured with real-time PCR. (B) Protein expression levels of N-cadherin, E-cadherin, α-SMA, and vimentin were assessed with Western blot assay. (C) Quantitation of Western blot signal intensities by ImageJ software. Data are expressed as mean ± SEM of five mice in each group. (*) p < 0.05, and (**) p < 0.01 versus vehicle-treated BLM model group (as control group), as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).
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