Erythropoietin Reduces Inflammation, Oxidative Stress, and Apoptosis in a Rat Model of Bleomycin-Induced Idiopathic Pulmonary Fibrosis

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial disease with unknown etiology and no effective cure, posing a great health burden to society. Erythropoietin (EPO) has been demonstrated to have protective roles in various tissues such as brain, spinal cord, heart, kidney and lung tissues. In this study, we investigate the specific anti-inflammatory, antioxidant and antiapoptotic effects of erythropoietin on lung tissue in a bleomycin-induced rat model of idiopathic pulmonary fibrosis.

Methods: Recombinant human EPO or saline was injected, and the animals were monitored for 14 days after bleomycin instillation. Their hematocrit and serum EPO levels were determined. Histological and immunohistochemical analyses were performed.

Results: The extent of tissue injury, determined through morphometric analysis, was significantly decreased in size in animals treated with erythropoietin. An immunohistochemical analysis of the expression of cyclooxygenase-2 (COX-2), inducible synthase of nitric oxide (i-NOS), metalloproteinase-9 (MMP-9), erythropoietin receptor (EPO-R), and cytochrome-C (cyt-C) found these enzymes to be decreased in a statistically significant manner in animals treated with erythropoietin when compared to a non-treated group.

Conclusions: The reduced expression of COX-2, i-NOS, MMP-9, EPO-R, and i-NOS in the lung tissues of animals treated with EPO indicates the anti-inflammatory, antioxidant and antiapoptotic action of erythropoietin, suggesting its potential therapeutic role in pulmonary fibrosis.

Keywords: anti-inflammatory; antiapoptotic; antioxidant; bleomycin; erythropoietin; idiopathic pulmonary fibrosis.

Figure 1

(A) Normal pulmonary parenchyma, absence of inflammation and fibrosis; (B) physiological architecture of alveoli and interstitial space; (C) milder pneumonitis; (D) massive accumulation of macrophages and diffuse bleeding (arrow); (E) fibrosis retreating and ventilation restoration; (F) severity of damage as a percentage (%) of the experimental animals per study group, classified on a 0–2 scale. * p < 0.001, with statistically significant differences between BLM/SAL and BLM/EPO groups.

Figure 2

Representative data of COX-2 expression: (A) BLM/SAL group, diffuse and strongly positive expression of the enzyme (arrow); (B) BLM/EPO group, less positive staining of the substrate with intense staining of the bronchus’ smooth muscle fibers (arrow).

Figure 3

Representative data of i-NOS expression: (A) BLM/SAL group, intense positive cells in the bronchial epithelium (arrow); (B) BLM/EPO group, resurfacing healthy cells and small foci of positive cells in the interstitial space (arrow).

Figure 4

Representative data of MMP-9 expression: (A) BLM/SAL group, diffuse positive fibroblasts in the interstitial and alveolar epithelium (arrows); (B) BLM/EPO group, mild positive staining in alveolar epithelial cells (arrow).

Figure 5

Representative data of EPO-R expression: (A) BLM/SAL group, multiple positive fibroblasts (arrow) flood the substrate and bronchial epithelium through freeing the lumen of the bronchi; (B) BLM/EPO group, mild positive staining in epithelial cells of the bronchus. No staining in the interstitial space.

Figure 6

Representative data of Cyt-c expression: (A) BLM/SAL group, diffuse seizure of the mid-alveolar space by positive fibroblasts (arrow); (B) BLM/EPO group, absence of positive staining areas throughout the field of vision, healthy cells.