Emodin ameliorates bleomycin-induced pulmonary fibrosis in rats by suppressing epithelial-mesenchymal transition and fibroblast activation

Abstract

Aberrant activation of TGF-β1 is frequently encountered and promotes epithelial-mesenchymal transition (EMT) and fibroblast activation in pulmonary fibrosis. The present study investigated whether emodin mediates its effect via suppressing TGF-β1-induced EMT and fibroblast activation in bleomycin (BLM)-induced pulmonary fibrosis in rats. Here, we found that emodin induced apoptosis and inhibited cellular proliferation, migration and differentiation in TGF-β1-stimulated human embryonic lung fibroblasts (HELFs). Emodin suppressed TGF-β1-induced EMT in a dose- and time-dependent manner in alveolar epithelial A549 cells. Emodin also inhibited TGF-β1-induced Smad2, Smad3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediated the emodin-induced effects on TGF-β1-induced EMT. Additionally, we provided in vivo evidence suggesting that emodin apparently alleviated BLM-induced pulmonary fibrosis and improved pulmonary function by inhibiting TGF-β1 signaling and subsequently repressing EMT, fibroblast activation and extracellular matrix (ECM) deposition. Taken together, our data suggest that emodin mediates its effects mainly via inhibition of EMT and fibroblast activation and thus has a potential for the treatment of pulmonary fibrosis.

Figure 1. Emodin alleviates bleomycin (BLM)-induced pulmonary fibrosis in rats.

Rats were intratracheally injected with a single dose of BLM (3.0 mg/kg) and subsequently received emodin (20 mg/kg) or vehicle by gavage daily for 21 days. (a) Representative images from different groups determined by Micro-CT showing radiologic features were presented. (b) Representative mimetic diagram drawn out from Micro-CT images, based on different tissues with varying density. (c) Lung tissue sections were prepared and stained with Masson’s trichrome staining (200x magnification). (d–f) Pulmonary function parameters including inspiratory resistance (Ri), expiratory resistance (Re) and pulmonary dynamic compliance (Cdyn) among different groups were compared three weeks after BLM instillation. Data are expressed as mean ± SD, n = 4, *P < 0.05; **P < 0.01.

Figure 2. Emodin inhibits cell proliferation, migration and differentiation in human embryonic lung fibroblasts (HELFs).

HELFs were treated with TGF-β1 (10 ng/mL) and emodin for 24 and 48 h, respectively. The cells treated with DMSO only served as control. (a,b) The viabilities of fibroblasts treated as indicated were measured by CCK-8 assay at 24 and 48 h, respectively. All the values were normalized to the control, representing 100% cell viability. (c) Wound healing assay was used to assess the effect of emodin on fibroblasts migration. The wound closure was photographed at post-scratching 24 and 48 h. Original magnification, ×400. (d,e) The wound closure rate at 24 and 48 h, representing the migration rate, was detected. (f) Immunofluorescence staining of α-SMA (a marker of myofibroblast differentiation) was performed. Original magnification, ×400. (g) Immunofluorescence of α-SMA was quantified by Image J software. Data are expressed as mean ± SD, n = 6, *P < 0.05; **P < 0.01.

Figure 3. Emodin promotes cell apoptosis in both basal and TGF-β1-stimulated human fibroblasts.

HELFs were treated with TGF-β1 (10 ng/mL) and the indicated concentrations of emodin (30 and 60 μM) for 24 h. (a) The cells were double-stained with Annexin V-FITC and PI, and then the cellular apoptosis was determined by flow cytometry. (b) The ratio of apoptotic cells (Annexin V+ PI-, Annexin V- PI+ and Annexin V+ PI+) was statistically analyzed. (c) The protein expression of caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, Bax and Bcl-2 was analyzed by Western blot assay. (d–f) Densitometric analysis of proteins of interest in the immunoblots using GAPDH as the internal reference. (g) The ratio of Bax/Bcl-2. Data are expressed as mean ± SD; n = 6, *P < 0.05; **P < 0.01.

Figure 4. Emodin suppresses TGF-β1-induced EMT in alveolar epithelial A549 cells.

A549 cells were treated with TGF-β1 (10 ng/mL) and different concentrations of emodin (15, 30, 60 μM) for 24 or 48 h. The cells stimulated with DMSO only served as controls. EMT was determined by examining (A) the morphological changes. Original magnification, ×400. (B,C) the expression levels of marker proteins of EMT including E-cadherin and fibronectin. (D,E) Quantitative densitometry of different samples using Western blot. Expression of E-cadherin and fibronectin was normalized to GAPDH level in the same sample. A549 cells were treated with TGF-β1 (10 ng/mL) and emodin (60 μM) for 24 h, and EMT was further examined by (F) immunofluorescence and (G) wound healing assay. (H) The wound closure rate, representing the migration rate, was detected. Data are expressed as mean ± SD, n = 6, *P < 0.05; **P < 0.01.

Figure 5. Emodin inhibits TGF-β1 signaling pathway in alveolar epithelial A549 cells.

A549 cells were treated as indicated for 24 or 48 h. (a,b) Western blot was used to analyze the phosphorylation levels of Smad2, Smad3 and Erk1/2. Results are representative of different experiments. (c,d) Scanning densitometry of western blot on different samples was analyzed quantitatively. Expression of p-Smad2, p-Smad3 and p-Erk1/2 was normalized to Smad2, Smad3 and Erk1/2 level, respectively. (e–g) Real-time PCR was performed to examine the mRNA level of TGF-β1, Slug and Snail. Data are expressed as mean ± SD, n = 6, *P < 0.05; **P < 0.01.

Figure 6. Emodin represses TGF-β1 expression, EMT and fibroblast activation in vivo.

Rats were intratracheally injected with a single dose of BLM (3.0 mg/kg) and followed by emodin (20 mg/kg) or vehicle by gavage daily for 21 days. (a) Western blot was used to analyze the protein levels of TGF-β1, FSP-1, α-SMA, E-cadherin and vimentin in the lung tissues. (b) Densitometric analysis of TGF-β1, FSP-1, α-SMA, E-cadherin and vimentin in the immunoblots using GAPDH as the internal reference. (c,d) Immunohistochemical staining of vimentin- and α-SMA-positive cells in the lungs, and images at 200× magnification. (e,f) Real-time PCR was performed to detect the mRNA levels of E-cadherin and vimentin in the pulmonary tissues derived from each group. Data are expressed as mean ± SD, n = 6, *P < 0.05; **P < 0.01.

Figure 7. Emodin reduces collagen production and ECM deposition in vivo.

Rats were intratracheally injected with a single dose of BLM (3.0 mg/kg) and followed by emodin (20 mg/kg) or vehicle by gavage daily for 21 days. (a) Hydroxyproline content in the lung tissues. (b–d) Real-time PCR was used to detect the mRNA levels of collagen I, collagen III, and TIMP-1 in the lung tissues. Data are expressed as mean ± SD, n = 6, *P < 0.05; **P < 0.01.