The Traditional Chinese Medicine Formula FTZ Protects against Cardiac Fibrosis by Suppressing the TGFβ1-Smad2/3 Pathway

Figures

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  Figure 1. FTZ improves cardiac dysfunction and alleviates cardiac hypertrophy in pressure overload-mediated cardiac dysfunction in mice (a) Representative M-mode echocardiography of the left ventricular chamber. FTZ-L and FTZ-H represent doses of 1.2 and 2.4 g/kg FTZ, respectively. CAPT represents the 10 mg/kg captopril group. (b–f) Echocardiographic assessment of LVIDd, LVIDs, EF%, FS%, and E/A; n = 8 per group. (g) HW/BW ratios in each group; n = 8 per group. Data are presented as the mean ± SEM. ∗∗P < 0.01 versus the sham group and #P < 0.05 and ##P < 0.01 versus the TAC group.

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  Figure 2. FTZ reduces cardiac fibrosis in pressure-overload mice. (a–d) H&E staining results of cardiac tissue are shown. Scale bars: 50 μm. n = 3 per group. Masson's trichrome staining results of cardiac tissue are shown. Scale bars: 50 μm. n = 3 per group. Fibrosis of cardiac tissues stained with Sirius Red. Scale bars: 50 μm. n = 3 per group. Immunohistochemical detection of α-SMA. Scale bars, 50 μm. n = 3 per group. (e–g) RT-qPCR to determine Col1A2, Col3, and CTGF expression. n = 6 per group. Data are presented as the mean ± SEM. ∗∗P < 0.01 versus the sham group and ##P < 0.01 versus the TAC group.

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  Figure 3. FTZ inhibits the proliferation and migration of cardiac fibroblasts in vitro. (a) Wound healing assay shows that FTZ intervention resulted in slow closing of scratch wounds after Ang-II treatment. Scale bars, 200 μm. n = 5 per group. (b) and (c) Representative images of EdU staining showing proliferating cells (stained red). Nuclei that are double-labeled with EdU (red) and Hoechst 33342 (blue) were considered to be new proliferative cells. The scale bar indicates 100 μm. n = 5 per group. (d) Effects of FTZ on cell viability were measured using a CCK-8 assay. n = 8 per group. Data are presented as the mean ± SEM. ∗∗P < 0.01 versus the control group and #P < 0.05 and ##P < 0.01 versus the Ang-II group.

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  Figure 4. FTZ reduces collagen synthesis in vitro. (a)–(d) RT-qPCR to determine the expression of Col1A2, Col3, CTGF, and α-SMA. n = 5 per group. (e)–(h) Col1A2, Col3, and α-SMA expression were determined using western blotting. n = 5 per group. Data are presented as the mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01 versus the control group and #P < 0.05 and ##P < 0.01 versus the Ang-II group.

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  Figure 5. FTZ improves cardiac fibrosis by inhibiting the TGFβ1-Smad2/3 pathway. (a) mRNA expression of TGFβ1 was determined using RT-qPCR. n = 5 per group. (b–e) Protein expression of TGFβ1, p-smad2, smad2, p-smad3, and smad3 were determined using western blotting. n = 5 per group. Data are presented as the mean ± SEM. ∗∗P < 0.01 versus the control group and ##P < 0.01 versus the Ang-II group. (f) and (g) TGFβ1 expression of consecutive sections of cardiac specimens assessed using immunohistochemical staining. Scale bar: 50 μm. n = 3 per group. Data are presented as the mean ± SEM. ∗∗P < 0.01 versus the sham group and ##P < 0.01 versus the TAC group.

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  Figure 6. Schematic diagram illustrating the mechanism of FTZ in regulating cardiac fibrosis.