Figure 1.
Changes in body weight in mice after administration of bleomycin.
Time course of changes in body weight in wild-type (WT) (n = 9) and in S1P3 knockout (KO) (n = 8) mice after administration of bleomycin. S1P3 KO mice exhibited decreased body weight loss compared to WT mice. The weight of the WT mice gradually decreased, reaching their lowest point on the seventh day after treatment and increasing afterwards. Values are presented as the mean ± SD (*p<0.05, **p<0.01).
Figure 2.
Histopathological findings of pulmonary inflammation.
Representative results of H&E staining of lung tissue from mice in the bleomycin-induced lung injury model (A, B, C and D). H&E staining of lung tissue from (A) wild-type (WT) and (B) knockout (KO) mice on the seventh day after intratracheal administration of bleomycin. H&E staining of lung tissue from (C) WT and (D) KO mice on the 28th day after intratracheal administration of bleomycin. All scale bars = 100 µm. The window shows an area of increased magnification revealing inflammatory cell infiltration. Representative results of H&E staining of lung tissue from mice in the saline-treated control group (E, F, G and H). H&E staining of lung tissue from (E) WT and (F) KO mice on the seventh day after intratracheal administration of saline. H&E staining of lung tissue from (G) WT and (H) KO mice on the 28th day after intratracheal administration of saline. All scale bars = 100 µm. The window shows an area of increased magnification revealing hyperplasia of the alveolar/bronchiolar epithelium.
Figure 3.
Histopathological findings of fibrosis induced by bleomycin.
Representative results of Masson's trichrome staining of lung tissue from mice in the bleomycin-induced lung injury model (A, B, C and D). Masson's trichrome staining of lung tissue from (A) wild-type (WT) and (B) knockout (KO) mice on the seventh day after intratracheal administration of bleomycin. Masson's trichrome staining of lung tissue from (C) WT and (D) KO mice on the 28th day after intratracheal administration of bleomycin. All scale bars = 100 µm. The window shows an area of increased magnification revealing a part of lung fibrosis. Representative results of Masson's trichrome staining of lung tissue from mice in the saline-treated control group (E, F, G and H). Masson's trichrome staining of lung tissue from (E) WT and (F) KO mice on the seventh day after intratracheal administration of saline. Masson's trichrome staining of lung tissue from (G) WT and (H) KO mice on the 28th day after intratracheal administration of saline.
Figure 4.
Ashcroft score of wild-type (WT) and S1P3 knockout (KO) mice on the seventh and 28th day after treatment with bleomycin or saline. No significant differences were observed between the Ashcroft scores of KO mice (2.72±0.30) and WT mice (2.96±0.23) on the seventh day after treatment with bleomycin. The Ashcroft scores of KO mice (3.56±0.39) were significantly lower than those of WT mice (5.72±0.68, p = 0.0006, n = 5 in each genotype) on the 28th day. In saline-treated control mice, almost no fibrosis of the lungs was observed on either the seventh day (0.13±0.09 vs. 0.07±0.09 in WT vs. KO mice, respectively; n = 3 in each genotype) or the 28th day (0.17±0.17 vs. 0.07±0.09 in WT vs. KO mice, respectively; n = 3 in each genotype).
Figure 5.
Total and differential white blood cell counts in bronchoalveolar lavage fluid (BALF).
A. Total cell count in BALF on the seventh day after intratracheal administration of bleomycin. Total cell counts in BALF collected on the seventh day after treatment were reduced by 56% in S1P3 knockout (KO) mice compared with wild-type (WT) mice (n = 5 in each genotype; **p<0.01). In saline-treated control mice, total cell counts in BALF collected on the seventh day after treatment were reduced by 25% in S1P3 KO mice compared with WT mice (n = 3 in each genotype, *p<0.05). Values are presented as the mean ± SD. B. Differential white blood cell counts in BALF collected after intratracheal administration of bleomycin or saline. The differential white blood cell counts from WT and KO mice had similar profiles (n = 5 in each genotype). Values are presented as the mean ± SD.
Figure 6.
Collagen levels in bronchoalveolar lavage fluid (BALF).
Collagen levels in BALF collected on the seventh and 28th day after intratracheal administration of bleomycin or saline. Knockout (KO) mice had significantly less pulmonary fibrosis compared with wild-type (WT) mice on the seventh day (n = 5 in each genotype, *p<0.05) and the 28th day (n = 5 in each genotype, **p<0.01). In saline-treated control mice, collagen levels were undetectable on both the seventh and 28th days.
Figure 7.
ELISA analyses of bronchoalveolar lavage fluid (BALF).
A. CTGF concentrations in BALF collected on the seventh day after intratracheal administration of bleomycin or saline. The CTGF concentrations in BALF after administration of bleomycin were significantly decreased in S1P3 knockout (KO) mice compared with wild-type (WT) mice (n = 5 in each genotype, *p<0.05). Values are presented as the mean ± SD. B. TGF-β1 concentrations in BALF collected on the seventh day after intratracheal administration of bleomycin or saline. No significant differences between WT and KO mice were observed in the concentration of TGF-β1 in BALF collected after administration of bleomycin (n = 5 in each genotype, p = 0.30). Values are presented as the mean ± SD. C. MCP-1 concentrations in BALF on the seventh day after intratracheal administration of bleomycin or saline. No significant differences between WT and KO mice were observed in the concentration of MCP-1 measured in BALF collected after administration of bleomycin (n = 5 in each genotype, p = 0.23). Values are presented as the mean ± SD.
Figure 8.
Concentration of S1P in bronchoalveolar lavage fluid (BALF).
S1P concentration in BALF collected on the seventh day after intratracheal administration of bleomycin or non-treated control. There were no significant differences in S1P concentration in BALF between wild-type (WT) and knockout (KO) mice at baseline or on the seventh day after bleomycin administration, nor were there significant differences in the S1P concentrations in BALF collected before and after bleomycin administration in WT (baseline vs. seventh day; p = 0.14) or KO (baseline vs. seventh day; p = 0.50) mice.