EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

Ying Ji Li,Hirofumi Inagaki,Toshiaki Sunazuka,Satoshi Omura,Kuniko Matsuda,Takako Shimizu,Satoru Takahashi,Arata Azuma,Shoji Kudoh,Tomoyuki Kawada,Yukiyo Hirata,Jiro Usuki,Shinji Abe

doi:10.1186/1465-9921-7-16

Abstract

BackgroundFourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.MethodsSeven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.ResultsBleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β.ConclusionThese findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

Highlights

Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects
We previously reported the preventive effects of 14-membered ring macrolides (14-MRMLs) in an animal experimental model of bleomycin-induced acute lung injury and subsequent fibrosis, which were mediated by anti-inflammatory mechanisms of action [5,6]
The increases in number of macrophages and Histopathologic assessment Bleomycin-induced pulmonary fibrosis was significantly inhibited by treatment with erythromycin 703 (EM703) on day 28 after bleomycin injection in ICRmice

Summary

Introduction

Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. Macrolides have been reported to improve the survival of patients with diffuse panbronchiolitis (DPB) and cystic fibrosis via anti-inflammatory effects [3,4]. We previously reported the preventive effects of 14-membered ring macrolides (14-MRMLs) in an animal experimental model of bleomycin-induced acute lung injury and subsequent fibrosis, which were mediated by anti-inflammatory mechanisms of action [5,6]. The pathogenesis of pulmonary fibrosis remains unclear, many investigators have found that neutrophil-mediated lung injury occurring in the acute inflammatory phase plays an important role in the progression of interstitial pneumonia [9,10,11]. TGF-β plays a key role in the development of idiopathic pulmonary fibrosis [1,12,13,14,15,16,17] and in experimental animal models of pulmonary fibrosis [18,19,20,21,22,23,24,25], and TGF-β intercellular signaling from the cell membrane to the nucleus occurs through Smad proteins [26]

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Results

Conclusion