Catalpol Attenuates Pulmonary Fibrosis by Inhibiting Ang II/AT1 and TGF-β/Smad-Mediated Epithelial Mesenchymal Transition

Figures

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  Figure 1. (A) Structure of catalpol (B) Animal experiment flowchart:C57BL/6 mice were treated by oral gavage with the different doses of CAT (100, 200 mg/kg/day), telmisartan (TEL) (10 mg/kg/day) and pirfenidone (PFD) (300 mg/kg/day) for 28 days followed by intratracheal instillation with BLM (5 mg/kg) or an equal volume of sterile normal saline (NS, Control group), respectively. Control group and BLM group received equal amounts of NS by oral gavage each day (n = 5-10, the number of mice sacrificed per group was 5, 5 and 10 on Day 7, 14 and 28 respectively). From left to right: Samples were collected at 7, 14 and 28th day after BLM administration. (C) Representative images of HE staining. Scale bar: 100 μm. (D) Representative images of Masson staining. Scale bar: 100 μm. (E–G) Expression of MMP2 and MMP9 in the lung tissues at 7, 14, 28d as detected by Western blot. (H,I) The MMP2 and MMP9 protein levels at 7, 14, 28d were analyzed by ImageJ software. (J) HYP in mouse serum at 7, 14, 28d was measured. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with the Control group, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the BLM group (ANOVA with Dunnett's post-hoc analysis).

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  Figure 2. Catalpol inhibits epithelial mesenchymal transition (EMT) in bleomycin induced pulmonary fibrosis. (A–C,J–L) Immunofluorescence analysis of E-cadherin in lung sections at 7, 14, 28d, E-cadherin (green), DAPI (blue), the fluorescence intensity of E-cadherin was analyzed by ImageJ software. (D–F,M–O) Immunofluorescence analysis of N-cadherin in lung sections at 7, 14, 28d, N-cadherin (red), DAPI (blue), the fluorescence intensity of N-cadherin was analyzed by ImageJ software. (G–I,P–R) Immunofluorescence analysis of α-SMA in lung sections at 7, 14, 28d, α-SMA (red), DAPI (blue), the fluorescence intensity of α-SMA was analyzed by ImageJ software. ###P < 0.001, ####P < 0.0001 compared with the Control group, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the BLM group (ANOVA with Dunnett's post-hoc analysis).

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  Figure 3. Catalpol inhibits the up-regulation of Ang II and AT1 in bleomycin-induced pulmonary fibrosis in mice. (A–D) Representative Western blots of AT1 and gray scale quantified data of AT1-to-β-actin expression ratio at 7, 14, 28d. (E) Ang II in mouse serum at 28 day was measured. ##P < 0.01, ####P < 0.0001 compared with the Control group, *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001 compared with the BLM group (ANOVA with Dunnett's post-hoc analysis).

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  Figure 4. Effect of catalpol on TGF-β/Smad2/3 signaling pathway. (A–C) Western blotting was conducted to detect the expression levels of TGF-β1, Smad2,3, p-Smad2, p-Smad3 and Snail in different lung tissue groups at 7, 14, 28d. (D–H) Densitometry data of TGF-β1-to-β-actin, p-Smad2-to-β-actin, p-Smad3-to-β-actin, Smad2/3-to-β-actin, Snail-to-β-actin expression ratio at 7, 14, 28d. ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with the Control group, *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001 compared with the BLM group (ANOVA with Dunnett's post-hoc analysis).

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  Figure 5. Schematic representation of catalpol regulation of the Ang II/AT1 and TGF-β/Smad pathway in pulmonary fibrosis.