Astragaloside IV modulates TGF-β1-dependent epithelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis

Abstract

Epithelial-mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti-fibrotic property in bleomycin (BLM)-induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM-induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM-induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM-induced EMT. Intriguing, transforming growth factor-β1 (TGF-β1) was found to be up-regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF-β1 and activated FOXO3a in lung tissues. TGF-β1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF-β1-activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down-regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF-β1-induced EMT. Moreover, ASV treatment, similar with the TGF-β1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF-β1/PI3K/Akt-induced FOXO3a hyperphosphorylation and down-regulation to reverse EMT during the progression of fibrosis.

Keywords: astragaloside IV; epithelial-mesenchymal transition; forkhead box O3a; idiopathic pulmonary fibrosis; transforming growth factor-β1.

Figure 1

Astragaloside IV‐treated mice are protected against bleomycin‐induced pulmonary fibrosis. Body weight (A) and lung wet‐to‐dry‐weight ratio (B) of each rat subjected to saline or bleomycin at 30 mL/kg for 28 d with or without 20 mg/kg Astragaloside IV treatment (n = 5/group). Representative micrographs (100×) with hematoxylin and eosin staining and Masson's trichrome staining of paraffin lung sections (C). The expression level of COLIA1 mRNA detected by qPCR (n = 5/group). Representative Western blot of Type I collagen protein and densitometrically quantified data of Type I collagen/GAPDH expression ratio. *P < .05 vs control; ^# P < .05 vs BLM

Figure 2

Inhibition of bleomycin‐induced oxidative stress and inflammation by the treatment with Astragaloside IV. Twenty‐eight days after bleomycin treatment, the markers of oxidative stress MDA (A), SOD (B), GSH‐PX (C), and the markers of inflammatory response TNF‐α (D) and IL‐6 (E) expression from the lungs of control group and those subjected to 30 mL/kg bleomycin with or without 20 mg/kg Astragaloside IV were measured. *P < .05 vs control; ^# P < .05 vs BLM

Figure 3

Astragaloside IV inhibited Bleomycin stimulated epithelial‐mesenchymal transition (EMT) in pulmonary fibrosis. A, Immunostaining of α‐SMA in lung tissue obtained from each group at 100 × magnification. B, Representative images of immunofluorescence double staining showing the overlap of E‐cadherin (red) and α‐SMA (green) accompanied with nuclei stained by DAPI (blue) in lung tissue sections. C, Expression of E‐cadherin and α‐SMA in the lung tissues as detected by Western blot analysis. *P < .05 vs control; ^# P < .05 vs BLM

Figure 4

Astragaloside IV suppressed Bleomycin induced upregulation of TGF‐β1 and hyperphosphorylation and inactivity of FoxO3a. A, relative expression of TGF‐β1 mRNA in groups as measured by qPCR. B, Representative Western blots of TGF‐β1 and densitometry quantified data of TGF‐β1‐to‐β‐actin expression ratio. C, relative expression of FoxO3a mRNA in each group. D, Immunocytochemistry staining of FoxO3a in bleomycin or saline‐treated rats with or without ASV. E, Representative Western blots and densitometry quantified data of p‐FOXO3a (Thr32), p‐FOXO3a (Ser253) and FOXO3a protein levels. β‐actin was used as the internal control. *P < .05 vs control; ^# P < .05 vs BLM

Figure 5

Astragaloside IV inhibited TGF‐β1 induced epithelial‐mesenchymal transition in pulmonary epithelial cells. A, TGF‐β1 treatment induces more elongated morphological shape and increases scattering, while addition of ASV suppressed these changes. B, Immunofluorescence double staining showing the overlap of E‐cadherin (red) and α‐SMA (green) in A549 cells in different groups. C, Western blots analysis of α‐SMA and E‐cadherin protein in each group. *P < .05 vs control; ^# P < .05 vs TGF‐β1

Figure 6

Astragaloside IV suppressed TGF‐β1/PI3K/Akt induced FOXO3a inactivity to reverse EMT in A549 cells. A, Western blots analysis of p‐FOXO3a (Thr32), p‐FOXO3a (Ser253), FOXO3a, p‐Akt and Akt protein levels. β‐actin was used as the internal control. B, Relative expression of FOXO3a mRNA level in A549. C, The expression levels of FOXO3a protein were tested by western blot analysis. D, Immunostaining of FOXO3a in A549 cells treated with 10 ng/mL TGF‐β1, 100 μg/mL ASV, 10 μmol/L SB431542 (a TGF‐β1 inhibitor) and or 20 μmol/L LY 294002 (a PI3K/Akt inhibitor). E, p‐FOXO3a (Thr32), p‐FOXO3a (Ser253), FOXO3a, p‐Akt and Akt expression via western blot following treatment with PBS, ASV, TGF‐β1, TGF‐β1 + ASV, TGF‐β1 + SB431542 or TGF‐β1 + LY 294002. F, Relative expression of FOXO3a mRNA in groups transfected with pcDNA‐3.1 + FOXO3a or pcDNA‐3.1 constructs. G, FOXO3a protein expression following transfection with a FOXO3a‐expressing plasmid. H, Western blots analysis of E‐cadherin and α‐SMA in indicated groups. I, Double‐labelled immunofluorescent staining was performed to examine the expression of E‐cadherin (red) and α‐SMA (green) in A549 cells in different groups. Scale bar = 50 μm *P < .05 vs control; ^# P < .05 vs TGF‐β1; ^$ P < .05 vs Vector

Figure 7

Astragaloside IV protected against bleomycin‐induced pulmonary fibrosis through suppressing TGF‐β1‐mediated EMT. Astragaloside IV inhibited TGF‐β1 induced activity of PI3K/Akt pathway independent inactivity of FOXO3a in pulmonary epithelial cell