Pretreatment with valproic acid alleviates pulmonary fibrosis through epithelial-mesenchymal transition inhibition in vitro and in vivo

Abstract

Epithelial-mesenchymal transition (EMT) plays a crucial role in the development of pulmonary fibrosis. This study aims to investigate the effects of valproic acid (VPA) on EMT in vitro and in vivo. In vitro, EMT was induced by the administration of transforming growth factor-β1 (TGF-β1) in a human alveolar epithelial cell line (A549). The dose effects of VPA (0.1-3 mM) on EMT were subsequently evaluated at different timepoints. VPA (1 mM) was applied prior to the administration of TGF-β1 and the expression of E-cadherin, vimentin, p-Smad2/3 and p-Akt was assessed. In addition, the effects of a TGF-β type I receptor inhibitor (A8301) and PI3K-Akt inhibitor (LY294002) on EMT were evaluated. In vivo, the effects of VPA on bleomycin-induced lung fibrosis were evaluated by assessing variables such as survival rate, body weight and histopathological changes, whilst the expression of E-cadherin and vimentin in lung tissue was also evaluated. A8301 and LY294002 were used to ascertain the cellular signaling pathways involved in this model. The administration of VPA prior to TGF-β1 in A549 cells prevented EMT in both a time- and concentration-dependent manner. Pretreatment with VPA downregulated the expression of both p-Smad2/3 and p-Akt. A8301 administration increased the expression of E-cadherin and reduced the expression of vimentin. LY294002 inhibited Akt phosphorylation induced by TGF-β1 but failed to prevent EMT. Pretreatment with VPA both increased the survival rate and prevented the loss of body weight in mice with pulmonary fibrosis. Interestingly, both VPA and A8301 prevented EMT and facilitated an improvement in lung structure. Overall, pretreatment with VPA attenuated the development of pulmonary fibrosis by inhibiting EMT in mice, which was associated with Smad2/3 deactivation but without Akt cellular signal involvement.

Fig. 1. VPA inhibits EMT in alveolar epithelial cells (A549) in a time- and concentration-dependent manner.

A A549 cells were treated with VPA (0–10 mM) for 48 h, and cell viability was assessed using an MTT assay. B A549 cells were treated with different concentrations of VPA with or without TGF-β1 (10 ng/ml) for 48 h and cell viability was assessed using an MTT assay. C The time-dependent effect of VPA: 1 mM VPA was administered 0.5 or 72 h prior to TGF-β1 and 0.5 h post TGF-β1, and cells were harvested 48 h after TGF-β1 administration and used for further analysis. D The expression of E-cadherin and vimentin and GAPDH serves as the loading control. E The concentration-dependent effect of VPA: VPA (0.1, 0.3, and 1 mM) was administered 72 h prior to TGF-β1 and cells were harvested 48 h after TGF-β1 administration and used for further analysis. F The expression of E-cadherin and vimentin in various conditions. Data are presented as dot plot and mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. Pretreatment with VPA inhibits EMT in alveolar epithelial cells.

A549 cells were treated with VPA (1 mM) 72  h prior to TGF-β1, and cells were collected 48  h after TGF-β1 stimulation. A The expression of E-cadherin and vimentin in various conditions, GAPDH serves as the loading control. B The expression of E-cadherin and vimentin in various conditions was analyzed by immunofluorescent staining. Mean fluorescence intensity was determined by Image J software. C The expression of p-Akt and p-Smad2/3 in various conditions. Data are presented as dot plot and mean ± SEM (n = 3). Scale bar = 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. VPA prevents EMT in alveolar epithelial cells induced by TGF-β1 and is dependent on Smad2/3 deactivation.

A549 cells were challenged with VPA (1 mM) 72 h prior to TGF-β1. Both LY294002 (20 μM) and A8301 (10 μM) were administered 0.5 h prior to TGF-β1, and cells were subsequently collected 48 h after TGF-β1 stimulation. A The expression of E-cadherin, vimentin, p-Akt, and p-Smad2/3 in various conditions. B The expression of E-cadherin and vimentin in various conditions was analyzed by immunofluorescent staining. Mean fluorescence intensity was determined by Image J software. Data are presented as dot plot and mean ± SEM (n = 3). Scale bar = 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. VPA and A8301 improve survival, reduce body weight loss, and mitigate lung fibrosis in mice.

Lung fibrosis was induced by endotracheal instillation of bleomycin (2 mg/kg). VPA (100 mg/kg) was administered intraperitoneally daily for 7 days before bleomycin instillation, followed by intraperitoneal administration daily for 28 days. LY294002 (5 mg/kg) or A8301 (1 mg/kg) were administered intraperitoneally once prior to bleomycin instillation. A Survival was analyzed by Kaplan–Meier test. *P < 0.05. B Change in body weight during the follow-up period. Data are presented as mean ± SEM. n = 10. *P < 0.05 compared with bleomycin-induced lung fibrosis. C H&E staining of lung slices. D Masson staining of lung slices. E Ashcroft score of lung tissue. Data are presented as dot plot and mean ± SEM (n = 5–6). Scale bar = 50 μm. *P < 0.05; **P < 0.01.

Fig. 5. VPA prevents EMT induced by bleomycin in fibrotic lung tissue.

A The expression of E-cadherin and vimentin in lung tissue from mice. B The expression of E-cadherin and vimentin in lung slices were analyzed by Immunofluorescent staining. Mean fluorescence intensity was determined by Image J software. Data are presented as dot plot and mean ± SEM (n = 5–6). Scale bar = 20 μm. *P < 0.05; **P < 0.01; ***P < 0.001.