Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

Abstract

Background: Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.

Methods: Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS-/-) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS-/- mice with BLM-induced pulmonary fibrosis.

Results: The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS-/- mice. Long-term treatment with the supplemental NO donor in n/i/eNOS-/- mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs.

Conclusions: These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.

Figure 1

Temporal changes in the body weight in a pulmonary fibrosis model at two weeks after BLM-treatment (n = 5-7). The changes in the ratios of the body weights at specific times/initial body weight from the start of BLM administration to the time of sacrifice (day 4, 10, 17 and 24) are shown. *P < 0.05 vs. BLM-treated WT mice.

Figure 2

The n/i/eNOS^−/−mice showed a deterioration of lung fibrosis in a pulmonary fibrosis model at two weeks after BLM-treatment. (A) Hematoxylin-eosin staining in normal saline (NS)-treated mice. Scale bar = 100 μm. (B) Hematoxylin-eosin staining, Masson-trichrome staining, α-SMA staining, MAC-2 staining in BLM-treated mice. Scale bar = 100 μm. (C) The fibrotic tissue area (blue-stained). (D) The collagen content in lung tissue. White and black bars indicate NS- (n= 3) and BLM- (n = 5) treated mice, respectively. *P < 0.05 vs. BLM-treated WT mice.

Figure 3

The n/i/eNOS^−/−mice showed an increase in the number of inflammatory cell in the bronchoalveolar lavage fluid in a pulmonary fibrosis model at two weeks after BLM-treatment. (A) The total cell counts. (B) The macrophage cell counts. (C) The lymphocyte cell counts. (D) The total protein concentrations. White and black bars indicate normal saline- (n = 3) and BLM-(n = 5) treated mice, respectively. *P < 0.05 vs. BLM-treated WT mice.

Figure 4

The n/i/eNOS^−/−mice were associated with an increase in the protein levels of proinflammatory cytokines, CC chemokine ligand 2 (CCL-2), and the tissue growth factor-β1 (TGF-β1) in a pulmonary fibrosis model at two weeks after BLM-treatment in BALF (n = 5). (A) IL-6 protein. (B) IL-1β protein. (C) TNF-α protein. (D) IFN-γ protein. (E) CCL-2 protein. (F) Active form of TGF-β1. *P < 0.05 vs. BLM-treated WT mice.

Figure 5

The mRNA expression of pro-inflammatory cytokines, CC chemokine ligand 2 (CCL-2), and the tissue growth factor-β1 (TGF-β1) in the lung in a pulmonary fibrosis model at two weeks after BLM-treatment (n = 5). (A) IL-6 mRNA expression. (B) IL-1β mRNA expression. (C) TNF-α mRNA expression. (D) IFN-γ mRNA expression. (E) CCL-2 mRNA expression. (F) TGF-β1 mRNA expression. *P < 0.05 vs. BLM-treated WT mice.

Figure 6

The n/i/eNOS^−/−mice exhibited an increased production of CTGF and collagen I. (A) Immunostaining for CTGF and collagen I in the lungs of the WT and n/i/eNOS^−/− mice. Scale bar = 100 μm. (B) CTGF mRNA expression (n = 5). (C) Collagen I mRNA expression (n = 5).

Figure 7

The anti-fibrotic effects of long-term treatment with a NO donor in a pulmonary fibrosis model at two weeks after BLM-treatment (n = 4-5). (A) Hematoxylin-eosin staining, Masson-trichrome staining. Scale bar = 100 μm. (B) The fibrotic tissue area (blue-stained). (C) The serum NOx levels. White and black bars indicate normal saline (NS)- and BLM- treated mice, respectively. (D) The collagen content in lung tissue. *P < 0.05 vs. the BLM-treated WT mice. ^#P < 0.05 vs. the BLM-treated n/i/e NOS^−/− mice that received ISDN. ^†P < 0.05 vs. the NS-treated mice.