Figure 2.

In vivo effects of HCl and mechanical ventilation on lung fibrosis and epithelial mesenchymal transition (EMT). (A) Experimental timeline of "two-hit" (HCl + MV) murine model. Mice were subjected to HCl stimulation followed by mechanical ventilation (MV) for 24 hours and 2 hours, respectively. (B) Hematoxylin and Eosin (HE) and Masson's trichrome staining of lung tissue (14d post-injury) showing inflammatory cell infiltration, alveolar wall thickening, and fibrotic cord formation. Blue: collagen. (C) Ashcroft fibrosis score in control (PBS), MV, and HCl-treated mice with or without MV. Data are presented as mean ± SEM. n = 4 mice /group. (D-F) Quantification of Collagen-I, hydroxyproline in lung tissue, and TGF-β1 levels in bronchoalveolar lavage fluid (BALF). Data are presented as mean ± SEM. n = 4 mice /group. (G) Immunofluorescence staining for E-cadherin (red) and Vimentin (green) in lung sections under different conditions. Nuclei were counterstained with DAPI (blue). (H-J) Western blot analysis of E-cadherin, Vimentin, and α-SMA protein expression in lung tissue under different conditions. (K-M) Quantification of E-cadherin (epithelial marker), Vimentin, and α-SMA (mesenchymal markers) expression levels normalized to GAPDH. Data are presented as mean ± SEM. n = 4 mice /group. (N) Immunofluorescence staining for PTEN (green) in lung sections under different conditions. Nuclei were counterstained with DAPI (blue). (O) Western blot analysis of PTEN protein expression in lung tissue under different conditions. (P) Quantification of PTEN expression levels normalized to GAPDH. Data are presented as mean ± SEM. n = 4 mice /group. One-way ANOVA and the Bonferroni post hoc test were used to compare more than two groups. Not significant (ns): P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.