Bellidifolin Ameliorates Isoprenaline-Induced Myocardial Fibrosis by Regulating TGF-β1/Smads and p38 Signaling and Preventing NR4A1 Cytoplasmic Localization

Figures

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  FIGURE 1. Chemical structure of Bellidifolin (BEL).

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  FIGURE 2. BEL ameliorates ISO-induced mouse myocardial fibrosis (A) HE staining to detect morphological changes; Masson trichrome staining to detect cardiac collagen deposition (B–D) Western blotting detection of collagen Ⅰ, collagen Ⅲ, and α-smooth muscle actin (α-SMA). Bar graphs show fold changes for the collagen Ⅰ, collagen Ⅲ, and α-SMA expression as analyzed by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, #p < 0.05 vs. ISO, ##p < 0.01 vs. ISO.

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  FIGURE 3. The effects of BEL on CFs proliferation in the presence or absence of TGF-β1 (A) The effects of different doses of BEL (7.5, 15, 30, 60, and 120 μM) on the proliferation of CFs induced by TGF-β1 using CCK-8 kit (B) The effects of different doses of BEL (7.5, 15, 30, 60, and 120 μM) on the viability of CFs without TGF-β1 stimulation using CCK-8 kit. Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. TGF-β1.

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  FIGURE 4. BEL decreases the expression of α-SMA, collagen Ⅰ, and collagen Ⅲ induced by TGF-β1 in CFs (A) The effects of different doses of BEL (15, 30, 60 μM) on α-SMA, collagen Ⅰ, and collagen Ⅲ expression induced by TGF-β1 using immunofluorescence staining. Cell nuclei was counterstained in blue with DAPI (B–D) Bar graphs show quantitative analyses of fluorescent signals for α-SMA, collagen I and collagen III (E–G) Western blotting detects the expression of α-SMA, collagen Ⅰ, and collagen Ⅲ expression. Bar graphs show fold changes of α-SMA, collagen Ⅰ, and collagen Ⅲ expression as analyzed by western blotting. GAPDH was used as a loading control (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, # p < 0.05 vs. TGF-β1, ## p < 0.01 vs. TGF-β1.

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  FIGURE 5. BEL suppresses the TβR phosphorylation induced by TGF-β1 in CFs (A and B) Western blotting detects TβRⅠ and TβRⅡ expression and phosphorylation level. Bar graphs show fold changes for the ratio of phosphorylated (P)-TβRI/TβRI, and P-TβRⅡ/TβRⅡ as analyzed by western blotting. GAPDH was used as a loading control (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, # p < 0.05 vs. TGF-β1, ## p < 0.01 vs. TGF-β1.

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  FIGURE 6. BEL limits the expression of TβRI and the phosphorylation of TβRI and II induced by ISO (A and B) Western blotting analysis of TβRII, P-TβRII, TβRI, and P-TβRI. Bar graphs show fold changes of P-TβRI/GAPDH, TβRI/GAPDH and P-TβRII/TβRII (n = 3). Data were shown as mean ± SEM. * p < 0.05 vs. control, **p < 0.01 vs. control, # p < 0.05 vs. ISO, ## p < 0.01 vs. ISO.

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  FIGURE 7. BEL inhibits Smads signaling in CFs (A) The effects of different doses of BEL (15, 30, 60 μM) on the expression of Smads2, 3, and 4 and the level of P-Smads2, 3 induced by TGF-β1 using immunofluorescence staining. Cell nuclei was counterstained in blue with DAPI (B–F) Bar graphs show quantitative analyses of fluorescent signals for P-Smads2, Smads2, P-Smads2, Smad3, and Smad4 (G–I) Western blotting detects Smad proteins expression and phosphorylation levels. Bar graphs show fold changes of P-Smad2/Smad2, P-Smad3/Smad3, and Smad4/GAPDH (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, # p < 0.05 vs. TGF-β1, ## p < 0.01 vs. TGF-β1.

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  FIGURE 8. BEL regulates Smad proteins expression and phosphorylation in vivo (A–C) Western blotting analysis of Smads2, 3, 4 expression and Smads2, 3 phosphorylation levels. Bar graphs show fold changes of P-Smad2/Smad2, P-Smad3/Smad3 and Smad4/GAPDH (n = 3). Data were shown as mean ± SEM. *p < 0.05 vs. control, **p < 0.01 vs. control, # p < 0.05 vs. ISO, ## p < 0.01 vs. ISO.

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  FIGURE 9. The effects of BEL on p38 signaling in vitro and in vivo (A) The effects of different doses of BEL (15, 30, 60 μM) on the expression p38 and the level of P-p38 in CFs induced by TGF-β1 using western blotting. Bar graphs show fold changes of P-p38/p38 (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. TGF-β1 (B) The effects of BEL on the expression p38 and the level of P-p38 in vivo by western blotting. Bar graphs show fold changes of P-p38/p38 (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. ISO.

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  FIGURE 10. BEL inhibits Smad3 phosphorylation independent of p38 in CFs (A) The expression of Smad3 and the level of P-Smad3 by western blotting. Bar graphs show fold changes of P-mad3/Smad3 (n = 3) (B–D) Expression of α-SMA, collagen Ⅰ, and collagen Ⅲ in CFs. Bar graphs show fold changes for the collagen Ⅰ, collagen Ⅲ, and α-SMA expression as analyzed by western blotting. GAPDH was used as a loading control (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. TGF-β1.

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  FIGURE 11. BEL impedes NR4A1 from the nucleus to cytoplasmic localization (A) Effects of BEL, SB203580 and LY2157299 on the expression of NR4A1 and P-NR4A1 induced by TGF-β1 using immunofluorescence staining. Cell nuclei was counterstained in blue with DAPI (B) Expression of NR4A1 and the levels of P-NR4A1 in total protein (T-NR4A1, T-P-NR4A1) by western blotting. Bar graphs show fold changes of P-NR4A1/NR4A1 (n = 3) (C) Expression of NR4A1 and the levels of P-NR4A1 in cytoplasmic protein (C-NR4A1, C-P-NR4A1) by western blotting. Bar graphs show fold changes of P-NR4A1/NR4A1 (n = 3) (D) Expression of NR4A1 and the levels of P-NR4A1 in nuclear protein (N-NR4A1, N-P-NR4A1) by western blotting. Bar graphs show fold changes of NR4A1/H3 (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, # p < 0.05 vs. TGF-β1, ## p < 0.01 vs. TGF-β1.

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  FIGURE 12. The proposed antifibrotic mechanism of BEL. BEL downregulates the phosphorylation of TβRI and TβRII, suppresses Smads2 and 4 expression and the phosphorylation and activation of Smads2 and 3 in CFs. Furthermore, BEL inhibits p38 phosphorylation and impedes NR4A1 cytoplasmic localization, which restores the inhibitory effect of NR4A1 to TGF-β1-profibrotic signaling. These inhibitory effects further caused a decrease in the expression of α-SMA, collagen I, and collagen III, thereby preventing proliferation and activation of CFs to alleviate myocardial fibrosis.