EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-beta signaling in lung fibroblasts.
BACKGROUND
Fourteen-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.


METHODS
Seven-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.


RESULTS
Bleomycin-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-beta). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-beta, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-beta.


CONCLUSIONS
These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-beta signaling in lung fibroblasts.
Li
Ying Ji
YJ
Fourth Department of Internal Medicine, Nippon Medical School, Tokyo, Japan. lyj@nms.ac.jp
Azuma
Arata
A
Usuki
Jiro
J
Abe
Shinji
S
Matsuda
Kuniko
K
Sunazuka
Toshiaki
T
Shimizu
Takako
T
Hirata
Yukiyo
Y
Inagaki
Hirofumi
H
Kawada
Tomoyuki
T
Takahashi
Satoru
S
Kudoh
Shoji
S
Omura
Satoshi
S
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001483:Base Sequence; D001761:Bleomycin; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D005347:Fibroblasts / Q000187:drug effects; D005786:Gene Expression Regulation; D006909:Hydroxyproline / Q000378:metabolism; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; C509870:EM703; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D016212:Transforming Growth Factor beta; D001761:Bleomycin; D004917:Erythromycin; D003094:Collagen; D006909:Hydroxyproline