Related Experiment Video
Updated: Jul 19, 2026
Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis
Published on: May 9, 2025
TIMP-1 is a key factor of fibrogenic response to bleomycin in mouse lung
B Manoury1, S Caulet-Maugendre, I Guénon
1INSERM U620, University of Rennes, France.
Abstract:
Pulmonary fibrosis is characterized by the excessive deposition of extracellular matrix in the interstitium, resulting in respiratory failure. The role of remodeling mediators such as metalloproteinases (MMPs) and their inhibitors (TIMPs) in the fibrogenic process remains misunderstood. We investigated MMP-9, MMP-2, TIMP-1, TIMP-2 and TIMP-3 in the fibrotic response to bleomycin of fibrosis prone C57BL/6J and fibrosis resistant BALB/c mice. Mice were administered with 0.1 mg bleomycin by intranasal administration. Either 24 h or 14 days after, the mice were anesthetized and underwent either bronchoalveolear lavage (BAL) or lung removal. Collagen deposition in lung tissue was determined by hydroxyproline measurement, MMP activity was analyzed by zymography, and other mediators were analyzed by ELISA. TIMP-1 was localized in lung sections by immunohistochemistry and real time PCR was performed to gene expression in lung. Non parametric Mann-Whitney and Spearman tests were used for statistical analysis. Fourteen days after bleomycin administration, hydroxyproline assay and histological study revealed that BALB/c mice developed significantly less fibrosis compared to C57BL/6J mice. At day 1, bleomycin enhanced TIMP-1, MMP-2 and MMP-9 protein levels in BALF, and induced corresponding genes in lung tissue of both strains. The rise of Timp-1, Mmp-9 and Mmp-2 gene levels were significantly stronger in lungs of C57BL/6J, whereas gelatinase activities of MMP-2 and MMP-9 were similar. Immunohistochemistry revealed that TIMP-1 macrophages and epithelial cells were prominent TIMP-1 producers in both strains. At day 14, neither MMP-2 nor MMP-9 levels exhibited strain-dependent protein level or gene expression, although TIMP-1 was strongly associated with fibrosis. Interestingly, bleomycin induced neither Timp-2 nor Timp-3 in lung tissue at any time of the study. The present study shows that early altered regulation of TIMP-1 following bleomycin administration may be involved in bleomycin-induced pulmonary fibrosis.
Insights
Altered regulation of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) early after bleomycin administration may contribute to pulmonary fibrosis development. This study highlights TIMP-1
Area of Science:
- Pulmonary Medicine
- Molecular Biology
- Toxicology
Background:
- Pulmonary fibrosis involves excessive extracellular matrix deposition, leading to respiratory failure.
- The roles of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in fibrosis are not fully understood.
- Investigating MMPs and TIMPs in bleomycin-induced pulmonary fibrosis models can elucidate fibrotic mechanisms.
Purpose of the Study:
- To investigate the roles of MMP-9, MMP-2, TIMP-1, TIMP-2, and TIMP-3 in the fibrotic response to bleomycin in fibrosis-prone (C57BL/6J) and fibrosis-resistant (BALB/c) mice.
- To determine the temporal and strain-dependent expression and activity of these mediators.
- To identify key cellular sources of TIMP-1 during bleomycin-induced lung injury.
Main Methods:
- Intranasal administration of bleomycin to C57BL/6J and BALB/c mice.
- Assessment of lung fibrosis via hydroxyproline measurement and histological analysis at 14 days post-bleomycin.
- Analysis of bronchoalveolar lavage fluid (BALF) and lung tissue for MMP and TIMP protein levels (ELISA, zymography) and gene expression (real-time PCR) at 24 hours and 14 days.
- Immunohistochemistry to localize TIMP-1 expression.
Main Results:
- BALB/c mice exhibited significantly less pulmonary fibrosis than C57BL/6J mice 14 days after bleomycin administration.
- Bleomycin increased TIMP-1, MMP-2, and MMP-9 protein levels and induced corresponding gene expression in both strains at day 1.
- Early increases in Timp-1, Mmp-9, and Mmp-2 gene expression were more pronounced in C57BL/6J mice, while gelatinase activities were similar.
- TIMP-1 was primarily localized in macrophages and epithelial cells.
- At day 14, MMP-2 and MMP-9 levels were not strain-dependent, but TIMP-1 strongly correlated with fibrosis.
- Timp-2 and Timp-3 were not induced by bleomycin in either strain.
Conclusions:
- Early altered regulation of TIMP-1 following bleomycin administration is implicated in the development of pulmonary fibrosis.
- TIMP-1, particularly its early upregulation, may play a critical role in mediating bleomycin-induced lung fibrosis.
- Further research is warranted to explore therapeutic strategies targeting TIMP-1 in pulmonary fibrosis.
