Effect of nilotinib on bleomycin-induced acute lung injury and pulmonary fibrosis in mice

Abstract

Background: The tyrosine kinase inhibitor imatinib mesylate was developed as an inhibitor of the kinase activity of BCR-ABL. However, imatinib also has potent inhibitory activity against the platelet-derived growth factor receptor (PDGFR). Nilotinib is approved for treating patients with chronic myeloid leukemia showing resistance or intolerance to imatinib. Like imatinib, nilotinib selectively inhibits the tyrosine kinase activity of PDGFR.

Objectives: We examined the effect of imatinib and nilotinib on acute lung injury and pulmonary fibrosis in a mouse model.

Methods: Mice were treated by intratracheal instillation of bleomycin. Imatinib or nilotinib were administered by oral gavage. To study the early inflammatory and late fibrotic phases of lung injury, mice were sacrificed on days 3, 7, 14 and 21 after bleomycin instillation.

Results: Histopathology showed that imatinib and nilotinib attenuated the extent of lung injury and fibrosis. The numbers of inflammatory cells and levels of IL-6, IL-1β and tumor necrosis factor-α were decreased in the imatinib and nilotinib groups on days 3 and 7. Imatinib and nilotinib therapy significantly reduced the levels of hydroxyproline on days 14 and 21, which was accompanied by decreased expression levels of transforming growth factor (TGF)-β1 and PDGFR-β. Imatinib and nilotinib also significantly reduced the expression levels of the genes for TGF-β1 and platelet-derived growth factor (PDGF). Imatinib and nilotinib treatment also significantly inhibited the PDGF-induced proliferation of lung fibroblasts in vitro. When imatinib or nilotinib was given 7 days after the instillation of bleomycin, only nilotinib attenuated pulmonary fibrosis.

Conclusions: Imatinib and nilotinib attenuated bleomycin-induced acute lung injury and pulmonary fibrosis in mice. In a therapeutic model, nilotinib showed more potent antifibrotic effects than imatinib.

Copyright © 2011 S. Karger AG, Basel.

Figures

Fig. 1

Representative histological lung sections from each group (R 200). Tissues were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin. Intratracheal administration of bleomycin induced lung injury with infiltration of inflammatory cells on days 3 and 7, and induced pulmonary fibrosis with deposition of collagen on days 14 and 21. These changes were attenuated by the treatment with imatinib or nilotinib. BLM = Bleomycin; BLM + I = bleomycin plus imatinib; BLM + N = bleomycin plus nilotinib; HE = hematoxylin and eosin; MT = Masson's trichrome.

Fig. 2

Quantitative examination of the effects of imatinib and nilotinib on bleomycin-induced lung injury (a) and pulmonary fibrosis (b). Compared with bleomycin treatment alone, the lung injury score was significantly lower in the imatinib or nilotinib treatment groups on day 7. The Ashcroft score was significantly lower in the imatinib treatment group on day 14 and in the nilotinib treatment group on days 14 and 21. The number of mice analyzed in each group ranged from 3 to 5. Abbreviations are as in figure 1. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N.

Fig. 3

Effects of imatinib and nilotinib treatments on inflammatory cytokines. The levels of IL-6 (a), IL-1β (b) and TNF-α (c) were measured in BAL fluid. Intratracheal administration of bleomycin induced increased levels of IL-6, IL-1β and TNF-α. Compared with the bleomycin treatment group, the level of IL-6 was significantly lower in the imatinib treatment group on days 3 and 7 and in the nilotinib treatment group on days 7, 14 and 21. The level of IL-1β was significantly lower in the imatinib treatment group on days 3 and 7 and in the nilotinib treatment group on day 7. The level of TNF-α was significantly lower in the nilotinib treatment group on day 7. The numbers of mice analyzed in each group ranged from 3 to 5. Abbreviations are as in figure 1. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N; ** p < 0.01 for BLM vs. BLM + I or for BLM vs. BLM + N.

Fig. 4

Effects of imatinib and nilotinib on the levels of hydroxyproline and TGF-β1. Compared with the bleomycin treatment group, the level of hydroxyproline was significantly lower in the imatinib treatment group on day 21 and in the nilotinib treatment group on days 7, 14 and 21. The level of TGF-β1 was significantly lower in the imatinib treatment group on days 3 and 21 and in the nilotinib treatment group on days 3, 14 and 21. The levels of hydroxyproline (a) and TGF-β1 (b) were measured in lung tissue. The number of mice analyzed in each group ranged from 3 to 5. Abbreviations are as in figure 1. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N; ** p < 0.01 for BLM vs. BLM + I or for BLM vs. BLM + N.

Fig. 5

Effects of imatinib and nilotinib treatments on the expression of TGF-β1 (a) and PDGFR-β (b). Compared with the bleomycin treatment group, the expression of TGF-β1 was significantly lower in the imatinib and nilotinib treatment groups on days 3, 7, 14 and 21. The expression of PDGFR-β was also significantly lower in the imatinib treatment group on days 3, 7 and 21 and in the nilotinib treatment group on days 3, 7, 14 and 21. Data are representative of 3 separate experiments. Abbreviations are as in figure 1, or as follows: Con = control; B + I = bleomycin plus imatinib; B + N = bleomycin plus nilotinib. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N; ** p < 0.01 for BLM vs. BLM + I or for BLM vs. BLM + N.

Fig. 6

Effects of imatinib and nilotinib treatments on the expression of pSmad2, pSmad3 and Smad7. Compared with the bleomycin treatment group, the expression of pSmad2 and pSmad3 was significantly lower in the nilotinib treatment group on days 14 and 21. The expression of Smad7 was significantly higher in the nilotinib treatment group on day 21. Data are representative of 3 separate experiments. Abbreviations are as in figures 1 and 5. * p

Fig. 7

Effects of imatinib and nilotinib treatments on the expression levels of genes encoding TGF-β1 (a) and PDGF (b). Compared with the bleomycin treatment group, the expression levels of the gene encoding TGF-β1 were significantly lower in the imatinib treatment group on days 14 and 21. The expression levels of the gene encoding PDGF were significantly lower in the imatinib treatment group on day 14 and in the nilotinib treatment group on days 14 and 21. Data are representative of 3 separate experiments. Abbreviations are as in figure 1. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N; ** p < 0.01 for BLM vs. BLM + I or for BLM vs. BLM + N.

Fig. 8

Effects of imatinib (a) and nilotinib (b) treatments on the growth of lung fibroblasts stimulated by PDGF. Compared with the PDGF-AA or PDGF-BB alone treatment groups, cell proliferation was significantly inhibited by the addition of imatinib or nilotinib. Data are representative of 3 separate experiments. * p < 0.05 for groups treated with PDGF-AA or PDGF-BB vs. groups treated with PDGF-AA or PDGF-BB plus imatinib or nilotinib; ** p < 0.01 for groups treated with PDGF-AA or PDGF-BB vs. groups treated with PDGF-AA or PDGF-BB plus imatinib or nilotinib.

Fig. 9

a Effects of imatinib and nilotinib treatments in a therapeutic model. Representative histological lung sections (×400) from each group. b Quantitative examination of the effects of imatinib and nilotinib on bleomycin-induced pulmonary fibrosis. c Results of BAL fluid analysis. Effect of imatinib and nilotinib on the levels of hydroxyproline (d) and TGF-β1 (e). Histopathology findings showed that nilotinib treatment attenuated bleomycin-induced pulmonary fibrosis, but imatinib treatment did not. Compared with the group treated with bleomycin alone, the Ashcroft score was significantly lower in the nilotinib treatment group, but not in the imatinib treatment group. Both imatinib and nilotinib treatments had little effect on the numbers of inflammatory cells in BAL fluid. Compared with the bleomycin treatment group, the levels of hydroxyproline and TGF-β1 were significantly lower in the nilotinib treatment group, but not in the imatinib treatment group. The number of mice analyzed in each group ranged from 3 to 8. Abbreviations are as in figure 1. * p < 0.05 for BLM vs. BLM + I or for BLM vs. BLM + N.