Anti-fibrotic Gene Therapy Effect of AP-1 Decoy ODN in Bleomycin-induced Sclerotic Animal Model

Song, Hyun Jeong; Lee, Kyu Suk

Abstract

BACKGROUND: Scleroderma is a connective tissue disorder characterized by excessive collagen production by activated fibroblasts. TGF-beta plays key roles in fibrosis of dermsis. Although numerous studies have elucidated the pathogenesis of scleroderma, effective therapeutic strategies for improving the sclerosis of the skin have been underinvestigated. Recently several studies indicated that an animal model of sclerotic skin induced by bleomycin is useful for providing clues and therapeutic interventions for scleroderma. We previously reported that AP (Activator protein)-1 decoy ODN (oligodeoxynucleotides) suppresses the TGF-beta1-induced type I collagen gene expression in cultured scleroderma fibroblast in vitro studies. Therefore, it is necessary to confirm the anti-fibrotic effect of AP-1 decoy ODN in sclerotic animal model.
OBJECTIVE
The purpose of this study is the establishment of a mouse model for scleroderma and confirmation of the anti-fibrotic effect of AP-l decoy ODN in vivo study.
METHODS
Dermal sclerosis was induced by intradermal injection of bleomycin at a dose of 0.3, 1.5, 3 (mg/ml) into the back skin of BALB/C mice twice a week for 4 weeks. To confirm anti-fibrotic effect of AP1-decoy ODN, the AP-1 decoy ODN was transfected into subcutaneous tissue of mice with or without bleomycin. Dermal sclerosis was examined by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. TGF-beta1 expression was detected by immunohistochemistry and type I collagen gene expression was also analyzed by dot blotting and western blot method.
RESULTS
Histopathological examination revealed that the dermal sclerosis with the deposition of thickened and homogenous collagen bundles increased according to the concentration of bleomycin. The expressions of type I collagen and TGF-beta1 were markedly increased in bleomyin-injected mice. Furthermore transfection of AP-1 decoy ODN with bleomycin suppressed the dermal sclerosis and type I collagen gene expression as well as TGF-beta1 in mice.
CONCLUSION
AP-1 decoy ODN inhibits the bleomycin-induced dermal sclerosis through down-regulation of type I collagen and TGF-beta1 expression in BALB/C mice. Thus the anti-fibrotic effect of AP-1 decoy ODN in bleomycin-induced sclerotic mouse model suggests the fundamental data for gene therapy of scleroderma.