Emodin Attenuates Bleomycin-Induced Pulmonary Fibrosis via Anti-Inflammatory and Anti-Oxidative Activities in Rats

Abstract

BACKGROUND Idiopathic pulmonary fibrosis (IPF) can severely damage lung function, which may result in death. Emodin is a major ingredient of rhubarb and has been proven to protect against lung disruptions. Our study focused on the potential medicinal effect of emodin against IPF. MATERIAL AND METHODS The experiment subjects were fully-grown male Sprague-Dawley rats with average weight of 180-220 kg. Histological analyses, Western blotting analysis, quantitative real-time PCR, and statistical analysis were used in the study. RESULTS We found that emodin significantly reduced lung structural distortion, collagen overproduction, massive inflammatory cells infiltration, proinflammatory cytokines expansion, and injuries caused by administration of bleomycin (BLM). Additionally, emodin suppressed the accumulation of p-IκBα and NF-κB, while stimulating the Nrf2-antioxidant signaling process in damaged lungs. Emodin inhibited epithelial-mesenchymal transition (EMT) induced by BLM in the lungs. Moreover, emodin suppressed the TGF-β1 expression and the downstream signal molecules p-Smad-2 and p-Smad-3, which are reinforced by BLM. Emodin can also reverse EMT-like shifts induced by recombinant TGF-β1 in alveolar epithelial cultured cells. CONCLUSIONS The effect of emodin in fibrotic lung injury is closely related to its favorable properties of anti-inflammation and anti-oxidation.

Figure 1

Emodin ameliorates BLM-triggered pulmonary fibrosis in rats. Rats received intratracheal injection of BLM and then were administrated with emodin 20 mg/kg each day by gavage for 21 days, with 5 rats per group. (A) Fractions of pulmonary tissue of control group, BLM group, and BLM + emodin group were stained with H&E. Scale bars 100 μm. (B) Extent of lung fibrosis was assessed and presented in percentages to the total area under evaluation. (C) Hydroxyproline levels of 3 groups were measured and compared with each other 3 weeks after BLM treatment. All data are presented as mean ±SD (n=5), * P<0.05; ** P<0.01.

Figure 2

Emodin suppressed inflammatory infiltration in BLM-induced pulmonary tissues. (A) Total number of cells and number of differentiate cell types in 1 mL BALFs was counted (n=5). (B) IL-1β, IL-6, and TNF-α level in cell-free BALFs supernatant were measured with ELISA kits. (C) The levels of protein expression of p-IκBα and nuclear translocation of NF-κB p65 were determined by Western blot analysis. (D) Quantitation of Western blot signal intensities. The study used endogenous GAPDH as a control for p-IκBα, and Histone H3 as a control for p65. All data are presented as mean ±SD (n=5), * P<0.05; ** P<0.01.

Figure 3

Emodin has anti-oxidative function and stimulates Nrf2 signaling in BLM-treated pulmonary tissues of rats. Activities of MDA, GSH (A), SOD, and GSH-Px (B) in control group, BLM group, and BLM + emodin group were measured using commercial kits. (C) Protein expression levels of nuclear Nrf2 and HO-1 were assessed by Western blot assay. (D) Quantitation of Western blot signal intensities. Histone H3 was used as a control for Nrf2 and GAPDH for HO-1. All data are presented as mean ±SD (n=5), ** P<0.01.

Figure 4

Emodin prohibits TGF-β1/Smad-2/-3 signaling transduction in BLM-damaged pulmonary tissue. (A) The mRNA expression levels of TGF-β1 were measured by qRT-PCR. (B) Protein expression levels of TGF-β1, smad2, p-smad2, smad3, and p-smad3 were evaluated with Western blot assay. (C) Quantitation of Western blot signal intensities. All data are presented as mean ±SD (n=5), ** P<0.01.

Figure 5

Emodin subdues BLM-induced EMT in rat pulmonary tissues. (A) Relative mRNA expression levels of indicate genes were measured with qRT-PCR. (B) Protein expression levels of E-cadherin, fibronectin, vimentin, and α-SMA were assessed with Western blot assay. (C) Quantitation of Western blot signal intensities. All data are presented as mean ±SD (n=5), ** P<0.01.

Figure 6

Emodin inhibits TGF-β1-induced EMT in A549 cells. The A549 cells were treated with TGF-β1 (10 ng/mL) and emodin (60 μM), while the cells treated with DMSO was set up as the control group. (A) Expression levels of E-cadherin, fibronectin vimentin, and α-SMA were measured by Western blot assay. (B) Quantitation of Western blot signal intensities. All data are expressed as mean ±SD from 3 independent experiments. ** P<0.01.