Non-Canonical Regulation of Type I Collagen through Promoter Binding of SOX2 and Its Contribution to Ameliorating Pulmonary Fibrosis by Butylidenephthalide

Figures

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  Figure 1. BP reduced collagen I production in lung fibroblast pre-stimulated with recombinant TGF-β1. TGF-β (5 ng/mL) stimulate for 12 h and (A) mRNA and (B) protein expression levels of BP treatment in several dosages (0, 10, 20, and 30 µg/mL) for 24 h on type I collagen, and SOX2 expressions. (C) mRNA and (D) protein expression levels of BP treatment for several time points (0, 1, 3, 6, 12, 24, and 48 h) in the dosage of 30 µg/mL on type I collagen and SOX2 expressions. The same amount of DMSO was added in 0 µg/mL groups as a vehicle control. Data showed 3 independent qPCR experiments and presented are mean ± SD. * denotes a significant decrease with the 0 µg/ml group of p < 0.05; ** p < 0.01; *** p < 0.001 by student’s t-test.

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  Figure 2. Effect of BP in canonical TGF-β downstream Smad and non-Smad pathways. Representative images of western blot analyses for TGF-β-induced (A) phosphorylated Smad2/3 for Smad pathway and its downstream EMT markers, and (B) phosphorylated Akt, p38 and JNK for non-Smad pathways and the effect of BP treatment.

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  Figure 3. Effects of exogenous overexpression of SOX2 on BP treatments in fibroblasts. Stable expression lines of pcDNA3.1/SOX2 and its vector control were isolated by G418 and treated with or without BP for 24 h. (A) Analysis of the Col I and SOX2 expressions were done on the mRNA levels by qPCR; and (B) analysis of the Col I, phospho-Smad2/3, and SOX2 expressions were done on the protein level by Western blot analysis (representative images). Data showed 3 independent qPCR experiments and presented are mean ± SD. * denotes a significant decrease with the - group of p < 0.05; *** p < 0.001 by student’s t-test.

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  Figure 4. Effects of BP and SOX2 on the activity of type I collagen promoter deletion constructs in NHLF. Luciferase reporter gene constructs including truncated sequences of the COL1A1 promoter and (A) Responsive elements exhibiting the previous described localization of consensus motifs of transcription factors SOX2, AP2, SP1, and TATA box are indicated. (B) The luciferase activity was determined 45 h after transfection of pGL3/COL1A1 in NHLF cells and BP treatment. Numbers represent the mean of 4 independent experiments. (C) ChIP analysis of SOX2 binding on the full length COL1A1 promoter in BP-treated groups, and CT values (upper) were obtained from qPCR analysis and the representative image (bottom) from three independent experiments of gel electrophoresis, respectively. * denotes a significant decrease with the full-length group of p < 0.05 by ANOVA followed by post hoc analysis of Dunnett’s test.

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  Figure 5. BP treatment in bleomycin-induced mice. (A) Schematic illustration experimental design. Mice were divided into four groups, and bleomycin was instilled on day 0 in all three experimental groups. The control group instilled the same volume of saline. Treatment groups were administered 0 (vehicle), 50 and 10 mg/kg doses of BP every other day for 30 days. Histological findings revealed lung inflammation and fibrosis by both (B) H&E (200 × magnification) and (C) Masson’s staining (100× magnification). (D) The Ashcroft score was measured with all field of slides, each view counted and represented with mean ± SEM from six mice per group. (E) The hydroxyl proline content was obtained from tissues of each lobes of lung. * denotes a significant decrease of p < 0.05 by ANOVA followed by post hoc analysis of Dunnett’s test.

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  Figure 6. BP reduces inflammatory response and expressions of collagen I and SOX2 in bleomycin-induced mice. BALF was isolated (n = 5) and analyzed by ELISA for changes in (A) IL-1β, (B) IL-6, (C) MIP1β, and (D) TNF-α. (E) Expressions of collagen I and SOX2 protein from lung tissues in mice (n = 3) were quantified by Western blot and standardized by β-Actin. * denotes a significant decrease of p < 0.05 by ANOVA followed by post hoc analysis of Dunnett’s test. Data presented are mean ± SD. (BALF, broncho alveolar lavage fluid; MIP-1β, Macrophage Inflammatory Proteins 1 beta.).

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  Figure 7. A short schematic mechanism of BP to treat pulmonary fibrosis. In a mouse model of bleomycin-induced pulmonary fibrosis, promoter binding enhances the collagen expression in fibroblast. BP inhibits SOX2 expression and its binding on collagen promoter blocks fibrogenesis in vitro and in vivo. ⊥: Inhibition of the gene expression; Gray upwards arrow ↑: Increased level of TGF-β.