Comparative study on mouse models of acute exacerbation of pulmonary fibrosis

Figures

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  Figure 1. Mouse models of AE-PF. (A) Flow chart of AE-PF mouse models. An intratracheal injection of BLM was performed on day 0, followed by a second intratracheal injection of ADV, LPS or an additional dose of BLM on day 14. (The mice were randomly divided into Ctrl group, BLM group and three AE groups. AE groups include three subgroups: BLM+ADV group, BLM+LPS group and BLM+BLM group. (B) The weight changes over 21 days. The weights of mice in three AE groups were significantly reduced in comparison with the BLM group at Day 21. (C) The survival of mice from day 14 to day 21 in BLM, BLM+ADV, BLM+LPS and BLM+BLM groups were 100% (12/12), 66.7% (8/12), 50.0% (6/12) and 66.7% (8/12), respectively. *P<0.05, **P<0.01, ***P<0.001. AE, acute exacerbation; Ctrl, control; BLM, bleomycin; ADV, replication-defective adenoviral vectors; LPS, lipopolysaccharide.

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  Figure 2. The chest imaging and pathology in AE-PF mouse models by intratracheally instillation with ADV, LPS and a second dose of BLM. (A) Chest micro-CT scans showed the lung injury area. (B) Modified Müller scoring system (33) CT lesion area scores (n=6). (C) Gross lung tissue specimens showed that the severity of lung injury was more exacerbated in three AE groups compared with BLM group. (D) H&E and Masson's trichrome staining of lung tissues indicated that the AE groups exhibited significantly more severe lung injury and fibrosis compared with the BLM group. (E) Ashcroft score of lung tissues in HE or Masson staining on day 21 (each group, n=6). (F) Quantification of blue-stained area percentage of lung tissues in Masson's trichrome staining (each group, n=6). *P<0.05, **P<0.01 and ****P<0.0001. Scale bar, 100 µm. AE, acute exacerbation; CT, computer tomography; Ctrl, control; BLM, bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide.

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  Figure 3. The pulmonary fibrosis of three AE group mice was significantly aggravated compared with the BLM group. (A) The hydroxyproline contents in both BLM+ADV and BLM+BLM groups of mice were higher than in the BLM+LPS group animals (each group, n=6). (B) Western blotting analysis revealed that the expressions of fibrosis markers Fibronectin, MMP-7, TGF-β1, Collagen I and α-SMA in the AE groups were significantly upregulated compared with the BLM group. (C) Semi-quantification of Fibronectin protein expression relative to GAPDH. (D) Semi-quantification of MMP-7 protein expression relative to GAPDH. (E) Semi-quantification of TGF-β1 protein expression relative to GAPDH. (F) Semi-quantification of Collagen I protein expression relative to GAPDH. (G) Semi-quantification of α-SMA protein expression relative to GAPDH (each group, n=6). The relative mRNA expressions of fibrosis markers (H) Fibronectin, (I) MMP-7, (J) TGF-β1, (K) Collagen I and (L) α-SMA in the AE groups were significantly increased compared with the BLM group by qPCR (each group, n=6). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 50 µm. AE, acute exacerbation; BLM, bleomycin; ADV, adenoviral vectors; LPS,…

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  Figure 4. The levels of inflammatory cytokines in BALF and serum were significantly elevated in AE-PF mice by ELISA. The levels of (A) IL-1β, (B) TNF-α and (C) IL-6 in BALF were significantly elevated in three AE-PF group mice compared with BLM group. The findings of the BLM group were significantly increased when compared with the Ctrl group (each group, n=6). The levels of (D) IL-1β, (E) TNF-α and (F) IL-6 in serum were significantly elevated in three AE-PF groups of mice compared with BLM group. The findings of the BLM group were significantly increased when compared with the Ctrl group (each group, n=6). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AE, acute exacerbation; BALF, bronchoalveolar lavage fluid; Ctrl, control; BLM, bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide.

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  Figure 5. Apoptosis was significantly enhanced in AE-PF mice, particularly in the BLM+ADV group. (A) Representative TUNEL staining images of lung tissues from each group. (B) Quantification of TUNEL-positive cells showed that the percentages in the AE groups were significantly higher than in the BLM group, particularly in the BLM+ADV group (each group, n=6). (C) Quantification of the Bax/Bcl2 ratio revealed a significant increase in the BLM+ADV group compared with both the BLM+LPS and BLM+BLM groups (each group, n=6). (D) Representative western blotting bands showing the protein expression of Bcl2 and Bax in each group. (E) The findings of quantitative-PCR analysis showed the Bax/Bcl2 ratio was significantly enhanced in three AE-PF group mice when compared with BLM group (each group, n=6). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar: 50 µm. AE, acute exacerbation; BLM, bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide.

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  Figure 6. Epithelial cell injury was significantly enhanced in the AE-PF mice. (A) Western blotting analysis of E-cadherin protein expression. (B) Quantification of E-cadherin expression relative to GAPDH (each group, n=6). (C) Representative immunofluorescence images of SPC staining. (D) Quantification of SPC fluorescence intensity (each group, n=6). *P<0.05, **P<0.01, ***P<0.001. Scale bar, 50 µm. AE, acute exacerbation; BLM, bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide; SPC, pro-surfactant protein C.