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Article Open Access

Comparative study on mouse models of acute exacerbation of pulmonary fibrosis

  • Authors:
    • Xu Ye
    • Mingrui Zhang
    • Mengying Liu
    • Huihui Zhu
    • Xiaoling Ye
    • Cheng Jiang
    • Qi Li
    • Xinmei Huang
    • Mengshu Cao
  • View Affiliations / Copyright

    Affiliations: Department of Respiratory and Critical Care Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, P.R. China, Department of Respiratory and Critical Care Medicine, Nanjing Drum Tower Hospital, Drum Tower Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210008, P.R. China
    Copyright: © Ye et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 253
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    Published online on: July 24, 2026
       https://doi.org/10.3892/etm.2026.13248
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Abstract

The etiology and underlying mechanisms of acute exacerbation of idiopathic pulmonary fibrosis (AE‑IPF) remain poorly understood. Although several animal models have been developed to study AE‑IPF, a systematic evaluation and comparison of these models has not yet been reported. In the present study, PF was induced in mice by a single intratracheal administration of bleomycin (BLM). On day 14 after the initial BLM challenge, AE‑PF in mice was induced by intratracheal re‑challenge with replication‑deficient adenoviral vectors (ADV), lipopolysaccharide (LPS) or a second dose of BLM. Micro‑chest computed tomography (CT) was performed on day 20, and blood, bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected after sacrifice on day 21. Compared with mice receiving a single dose of BLM alone, all three AE‑PF groups exhibited significant body weight loss and increased mortality (with the highest mortality in the LPS group), as well as more extensive lung injury on CT. Histopathological scores for inflammation and fibrosis, hydroxyproline content and expression levels of fibrotic markers (fibronectin, collagen I, α‑smooth muscle actin and MMP7) were significantly elevated in the AE‑PF groups. Inflammatory cytokines (IL‑6, IL‑1β and TNF‑α) were markedly increased in both serum and BALF. Furthermore, the AE‑PF models showed downregulation of alveolar epithelial cell markers (E‑cadherin and pro‑surfactant protein C) and a significant increase in apoptotic activity. Notably, fibrosis progression was more severe in the ADV and two‑dose BLM groups than in the LPS group. Taken together, these findings indicate that all three triggers can induce AE‑PF through enhanced apoptosis, inflammation and fibrosis. These results support the use of day 14 re‑challenge as a standardized model for AE‑PF. Among the three models, the ADV‑induced AE‑PF model may serve as a particularly suitable platform for investigating the pathogenesis of acute exacerbations in idiopathic pulmonary fibrosis.
View Figures

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.

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.

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, lipopolysaccharide; α-SMA: α-smooth muscle actin.

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.

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.

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.
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Copy and paste a formatted citation
Spandidos Publications style
Ye X, Zhang M, Liu M, Zhu H, Ye X, Jiang C, Li Q, Huang X and Cao M: Comparative study on mouse models of acute exacerbation of pulmonary fibrosis. Exp Ther Med 32: 253, 2026.
APA
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C. ... Cao, M. (2026). Comparative study on mouse models of acute exacerbation of pulmonary fibrosis. Experimental and Therapeutic Medicine, 32, 253. https://doi.org/10.3892/etm.2026.13248
MLA
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C., Li, Q., Huang, X., Cao, M."Comparative study on mouse models of acute exacerbation of pulmonary fibrosis". Experimental and Therapeutic Medicine 32.4 (2026): 253.
Chicago
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C., Li, Q., Huang, X., Cao, M."Comparative study on mouse models of acute exacerbation of pulmonary fibrosis". Experimental and Therapeutic Medicine 32, no. 4 (2026): 253. https://doi.org/10.3892/etm.2026.13248
Copy and paste a formatted citation
x
Spandidos Publications style
Ye X, Zhang M, Liu M, Zhu H, Ye X, Jiang C, Li Q, Huang X and Cao M: Comparative study on mouse models of acute exacerbation of pulmonary fibrosis. Exp Ther Med 32: 253, 2026.
APA
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C. ... Cao, M. (2026). Comparative study on mouse models of acute exacerbation of pulmonary fibrosis. Experimental and Therapeutic Medicine, 32, 253. https://doi.org/10.3892/etm.2026.13248
MLA
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C., Li, Q., Huang, X., Cao, M."Comparative study on mouse models of acute exacerbation of pulmonary fibrosis". Experimental and Therapeutic Medicine 32.4 (2026): 253.
Chicago
Ye, X., Zhang, M., Liu, M., Zhu, H., Ye, X., Jiang, C., Li, Q., Huang, X., Cao, M."Comparative study on mouse models of acute exacerbation of pulmonary fibrosis". Experimental and Therapeutic Medicine 32, no. 4 (2026): 253. https://doi.org/10.3892/etm.2026.13248
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