Matrine suppresses cardiac fibrosis by inhibiting the TGF‑β/Smad pathway in experimental diabetic cardiomyopathy

Abstract

Cardiac fibrosis is one of the pathological characteristics of diabetic cardiomyopathy (DbCM). Matrine treatment has proven to be effective in cases of organ fibrosis and cardiovascular diseases. In the present study, the anti-fibrosis-associated cardioprotective effects of matrine on DbCM were investigated. Rats with experimental DbCM were administered matrine orally. Cardiac functions were evaluated using invasive hemodynamic examinations. Cardiac compliance was assessed in isolated hearts. Using Sirius Red and fluorescence staining, the collagen in diabetic hearts was visualized. MTT assay was used to select non‑cytotoxic concentrations of matrine, which were subsequently used to treat isolated cardiac fibroblasts incubated under various conditions. Western blotting was performed to assess activation of the transforming growth factor‑β1 (TGF‑β1)/Smad signaling pathway. Rats with DbCM exhibited impaired heart compliance and left ventricular (LV) functions. Excessive collagen deposition in cardiac tissue was also observed. Furthermore, TGF‑β1/R‑Smad (Smad2/3) signaling was revealed to be markedly activated; however, the expression of inhibitory Smad (I‑Smad, also termed Smad7) was reduced in DbCM. Matrine administration led to a marked recovery in LV function and heart compliance by exerting inhibitory effects on TGF‑β1/R‑Smad signaling pathway‑induced fibrosis without affecting I‑Smad. Incubation with a high concentration of glucose triggered the TGF‑β1/R‑Smad (Smad2/3) signaling pathway and suppressed I‑Smad signaling transduction in cultured cardiac fibroblasts, which led to an increase in the synthesis of collagen. After cardiac fibroblasts had been treated with matrine at non‑cytotoxic concentrations without affecting I‑Smad, matrine blocked TGF‑β1/R‑Smad signaling transduction to repress collagen production and deposition. In conclusion, the results of the present study demonstrated that TGF‑β1/Smad signaling‑associated cardiac fibrosis is involved in the impairment of heart compliance and LV dysfunction in DbCM. By exerting therapeutic effects against cardiac fibrosis via its influence on TGF‑β1/Smad signaling, matrine exhibited cardioprotective effects in DbCM.

Figure 1.

Cardiac compliance detection and plotting in isolated hearts. The line chart in this figure demonstrates the P-V (left ventricular diastolic pressure-left ventricular volume) curve of isolated hearts from the Ctrl, Mat, DbCM and DbCM+Mat groups. Results are presented as the mean ± standard deviation. Approximately linear trends are shown. *P<0.05 vs. Ctrl; **P<0.05 vs. Mat; ***P<0.05 vs. DbCM. Ctrl, control; Mat, matrine; DbCM, diabetic cardiomyopathy.

Figure 2.

Collagen deposition and TGF-β1/Smad signaling pathway activation in cardiac tissue. (A) In the left part of the figure, captured images of Sirius Red staining of cardiac tissue harvested from each group (Ctrl, Mat, DbCM and DbCM+Mat) are shown. The bar chart on the right shows the values of the optical densities of Sirius Red in each group (results are presented as the mean ± standard deviation). (B) Immunoblots of TGF-β1, Smad2, phospho (p)-Smad2, Smad3, p-Smad3, Smad7, collagen I and the loading control, GAPDH, are shown. The bar chart on the right shows the relative expression levels of TGF-β1 (TGF-β1/GAPDH), Smad7 (Smad7/GAPDH), phosphorylated Smad2 (phospho-Smad2/Smad2), phosphorylated Smad3 (phospho-Smad3/Smad3) and collagen I (collagen I/GAPDH). *P<0.05 vs. Ctrl; **P<0.05 vs. Mat; ***P<0.05 vs. DbCM. TGF-β1, transforming growth factor-β1. Ctrl, control; Mat, matrine; DbCM, diabetic cardiomyopathy.

Figure 3.

Cell viability of isolated CFs incubated with matrine at different concentrations. This figure demonstrates the results of the MTT assay, which determined the cell viability rate (%) of isolated CFs incubated with matrine at concentrations of 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mmol/l. *P<0.05 vs. the previous concentration in the series. CF, cardiac fibroblast.

Figure 4.

TGF-β1/Smad signaling pathway activation in isolated cardiac fibroblasts. (A) Immunoblots of TGF-β1, Smad2, phospho (p)-Smad2, Smad3, p-Smad3, Smad7 and GAPDH in isolated cardiac fibroblasts treated with serial concentrations of matrine (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mmol/l) and/or HG medium are shown. (B) The bar chart shows the relative expression levels of TGF-β1 (TGF-β1/GAPDH). (C) Bar charts also show the phosphorylation of Smad2 (p-Smad2/Smad2), (D) the phosphorylation of Smad3 (p-Smad3/Smad3), (E) the relative expression level of Smad7 (Smad7/GAPDH), and (F) the relative expression level of collagen I (collagen I/GAPDH). Data shown in B-F represent the mean ± standard deviation. *P<0.05 vs. first column; **P<0.05 vs. third column; ***P<0.05 vs. fourth column; ^#P<0.05 vs. fifth column. TGF-β1, transforming growth factor-β1.