Bellidifolin Inhibits SRY-Related High Mobility Group-Box Gene 9 to Block TGF-β Signalling Activation to Ameliorate Myocardial Fibrosis

Figures

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  Figure 1. BEL did not affect HCF cells viability. HCFs were administered with different doses of BEL (3.125, 6.25, 12.5, 25, 50, and 100 μM) for 24 h, and a CCK-8 assay was applied to investigate the cell viability.

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  Figure 2. BEL prevented TGF-β1-increased the expression of α-SMA, Collagen I, and Collagen III. (a) Western blot examined the expression of α-SMA, Collagen I, and Collagen III. (b–d) The fold changes of the Western blot results for α-SMA, Collagen I, and Collagen III were analysed; GAPDH was used as a loading control (n = 3). Data were presented as the mean ± standard error of the mean (SEM). ∗p < 0.05 and ∗∗p < 0.01 vs. Control; ##p < 0.01 vs. TGF-β1.

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  Figure 3. BEL reduced SOX9 expression and Smad3 phosphorylation. (a, c) TGF-β1-stimulated the expression of SOX9 and Smad3 and the level of Smad3 phosphorylation were analysed by Western blot. (b, d) Bar graphs presented fold changes of SOX9 and P-Smad3/Smad3. GAPDH was used as a loading control (n = 3). Data were presented as mean ± SEM. ∗∗p < 0.01 vs. Control; #p < 0.05 and ##p < 0.01 vs. TGF-β1.

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  Figure 4. SOX9 knockdown suppressed TGF-β1/Samd3 pathway activation. (a, c, e) Western blot and qRT-PCR evaluated siRNA transfection efficiency, and bar graphs presented fold changes of SOX9 expression. GAPDH was used as a loading control (n = 3). (b, d, f) Western blot and qRT-PCR analysed the effect of SOX9-Homo-905 siRNA on SOX9 administrated by TGF-β1. (g, h) The effect of knockdown of SOX9 on the expression of TGF-β1 and Smad3 and the level of Smad3 phosphorylation in the presence of TGF-β1 were detected by western blot. (i, j) The fold changes of TGF-β1 and P-Smad3/Smad were presented by bar graphs. GAPDH was used as a loading control (n = 3). Data were presented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 vs. Control + NC; #p < 0.05 vs. TGF-β1 + NC.

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  Figure 5. SOX9 knockdown inhibited TGF-β1-induced the expression of α-SMA, Collagen I, and Collagen III. (a–c) The expression of α-SMA, Collagen I, and Collagen III were assessed by Western blot. (d–f) Bar graphs presented fold changes of α-SMA, Collagen I, and Collagen III. GAPDH was used as a loading control (n = 3). Data were presented as the mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 vs. Control + NC; #p < 0.05 vs. TGF-β1 + NC.

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  Figure 6. BEL ameliorated ISO-induced morphological changes in mice. (a) The flow diagram for the experimental design in mice. (b) HE and Masson staining examined morphological changes in mice. (c, d) Bar graphs showed the area of myocardial injury and the percentage of collagen deposition. ∗∗p < 0.01 vs. Control; ##p < 0.01 vs. ISO.

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  Figure 7. BEL prevented ISO-induced elevations in Collagen I, Collagen III, α-SMA, and SOX9 in mice. (a) Immunohistochemical staining examined the expression of Collagen I and Collagen III. (b) Western blot analysed the expression of α-SMA, Collagen I, and Collagen III. (c–e) The fold changes of α-SMA, Collagen I, and Collagen III were shown by bar graphs; GAPDH was used as a loading control (n = 3). (f) Detecting the expression of SOX9 using Western blot. (g) The fold changes of SOX9 were presented by bar graphs. GAPDH was used as a loading control (n = 3). Data were presented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 vs. Control; #p < 0.05 and ##p < 0.01 vs. ISO.

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  Figure 8. The proposed mechanism of BEL on myocardial fibrosis.