Tangshen Formula Improves Diabetes-Associated Myocardial Fibrosis by Inhibiting TGF-β/Smads and Wnt/β-Catenin Pathways

Figures

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  Figure 1. Pathologic changes after Tangshen Formula (TSF) and irbesartan treatments. (A) Body weights were recorded once a week. Data are expressed as mean ± SEM. ###p < 0.001, ####p < 0.0001 KKAy group vs. C57BL/6J group. (B) Fasting blood glucose. After 16 weeks, glucose levels in KKAy mice were significantly increased. TSF-treated KKAy mice exhibited no statistical differences in glucose levels. Data are expressed as mean ± SEM. ###p < 0.001, ####p < 0.0001 KKAy group vs. C57BL/6J group. (C) Histology (Masson trichrome staining, original magnification, 200×) and semiquantification of the collagen area. Data are expressed as mean ± SEM. n = 6. *p < 0.05 TSF-treated group vs. KKAy group; ###p < 0.001 ####p < 0.0001 KKAy group vs. C57BL/6J group. (D) Ultrastructural findings of myocardial tissues in four groups of mice. Magnification ×12,000. Scale bar = 1 μm. Black arrows indicate mitochondria. Blue arrows indicate mitochondria vacuolation.

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  Figure 2. Tangshen Formula inhibited myocardial fibrosis in KKAy mice. (A) Immunohistochemistry (IHC) of collagen III (ColIII, original magnification, 200×), collagen I (ColI, original magnification, 200×), and alpha smooth muscle actin (α-SMA, original magnification, 200×). (B) IHC and semi-quantitative analyses for collagens I and III, and α-SMA. Data are expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01 TSF-treated group vs. KKAy group; #p < 0.05, ##p < 0.01, ###p < 0.001 KKAy group vs. C57BL/6J group. (C) Western blot and semi-quantitative analyses of ColI, ColIII, and α-SMA expressions.

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  Figure 3. Tangshen Formula treatment inhibited the expression of transforming growth factor-β1 (TGF-β1) and p-Smad2/3 and promoted the expression of smad7 in KKAy mice. (A) Western blot and semi-quantitative analyses of TGF-β1, smad7, p-Smad2/3, and Smad2/3 expressions. (B) IHC of psmad2/3 (original magnification, 400×) and the number of positive cells in each group. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001 TSF-treated group vs. KKAy group; ##p < 0.01, ####p < 0.0001 KKAy group vs. C57BL/6J group.

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  Figure 4. An effect of TSF on the canonical Wnt pathway. (A) IHC of anti-β-catenin (original magnification, 200×). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 TSF-treated group vs. KKAy group; #p < 0.05, ##p < 0.01, ###p < 0.001 KKAy group vs. C57BL/6J group. (B) Western blot analyses and semi-quantitative analyses revealed expressions of Wnt1, active-β-catenin, β-catenin, FN, and MMP-7 in KKAy mice and the different treatment groups. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 KKAy group vs. C57BL/6J group. *p < 0.05, **p < 0.01 TSF-treated group vs. KKAy group.

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  Figure 5. An inhibitory effect of TSF against the activation of the TGF-β/smad pathway in vitro. (A) The expression of fibrosis index protein in cardiac fibroblast (CF) cells stimulated by TGF-β in different concentrations at different times. (B) Quantitative analyses of protein expressions. (C) Quantificational real-time polymerase chain reaction (qRT-PCR) of messenger RNA (mRNA) expressions of fibrosis index of CFs induced by TGF-β1. (D) Effects of different concentrations of TSF on the survival rate of cells. (E) qRT-PCR of mRNA expressions of collagens I and III and α-SMA in CFs induced by TGF-β1 in the different groups. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 6. Schema of the possible mechanism of antifibrosis action of TSF. In the TGF-β/smad pathway, TSF decreased the expression of TGF-β1, inhibited the phosphorylation of Smad2/3, and increased the expression of Smad7, which promoted the degradation of catenin. Meanwhile, TSF also inhibited the Wnt pathway and further promoted β-catenin phosphorylation and degradation. The green arrow represents the effect of TSF.