Atorvastatin attenuates TGF‑β1‑induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

Figures

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  Figure 1. Effect of ATV on TGF-β1-induced proliferation of hVFs. (A) Cells were exposed to different concentrations of ATV, and then the viability of the cells was assessed using a CCK-8 assay. (B) hVFs were pretreated with various concentrations of ATV (0, 2, 5 and 10 µM) for 3 h, and then exposed to 5 ng/ml TGF-β1 for 24 h. The proliferation of hVFs was then assessed by CCK-8 assay. All data are presented as the mean ± standard error of mean of three independent experiments and described as a percentage of the control group. *P<0.05, **P<0.01 vs. untreated control group. #P<0.05 vs. TGF-β1 only-treated group. hVFs, human ventricular fibroblasts; ATV, atorvastatin; TGF-β1, transforming growth factor-β1; NS, not significant; CCK-8, Cell Counting Kit-8.

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  Figure 2. ATV inhibits the trans-differentiation of hVFs into myofibroblasts. Cells were exposed to ATV (10 µM) for 3 h and then co-treated with 5 ng/ml TGF-β1 for a further 24 h. (A) Cells were then immunostained with anti-α-SMA antibody (green) and stained with DAPI (nuclei; blue). Magnification, ×200. (B) The α-SMA mRNA level was assessed by reverse transcription-quantitative PCR assay. (C) The protein expression of α-SMA was measured by western blotting. (D) Quantitative analysis of α-SMA protein level in hVFs. The relative value was normalized to GAPDH expression. The data are presented as the mean ± standard error of mean of three independent experiments. **P<0.01, ***P<0.001 vs. untreated control group. #P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; hVFs, human ventricular fibroblasts; α-SMA, α-smooth muscle actin.

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  Figure 3. ATV inhibits TGF-β1-stimulated ECM production in hVFs. Fibroblasts were pretreated with ATV (10 µM) for 3 h and then co-stimulated with 5 ng/ml TGF-β1 for a further 24 h. (A) The mRNA levels of MMP-2, type I collagen and type III collagen were assessed by reverse transcription-quantitative PCR. (B) The protein expression levels of MMP-2, collagen I and collagen III were detected by western blotting. (C) Quantitative analysis of the western blot results was performed by evaluating the protein band densities with Image Pro-Plus version 6.0 software. The relative value was normalized to GAPDH expression. All data are expressed as the mean ± standard error of mean of three independent experiments. **P<0.01, ***P<0.001 vs. untreated control group. #P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; ECM, extracellular matrix; hVFs, human ventricular fibroblasts; MMP-2, matrix metalloproteinase-2.

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  Figure 4. ATV inhibits TGF-β1-induced Smad3 and MAPK signaling activation in hVFs. Fibroblasts were pre-incubated with ATV (10 µM) for 3 h and then co-treated with 5 ng/ml TGF-β1 for 30 min. The protein levels of (A) p-Smad3 and t-Smad3, (B) p-ERK1/2 and t-ERK1/2, (C) p-p38 and t-p38, and (D) p-JNK and t-JNK were detected using western blotting. The relative density was expressed as the ratio of p-protein to the corresponding t-protein. Values represent the mean ± standard error of mean of three independent experiments. **P<0.01, ***P<0.001 vs. untreated control group. #P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; MAPK, mitogen-activated protein kinase; hVFs, human ventricular fibroblasts; p-, phosphorylated; t-, total; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase.

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  Figure 5. Schematic diagram demonstrating how atorvastatin inhibits TGF-β1-induced fibrogenesis in hVFs. TGF-β1 treatment can induce activation of the Smad3 and MAPK signaling pathways, cause an increase in ECM synthesis and promote the fibrogenesis in hVFs. Atorvastatin plays a protective role and can prevent TGF-β1-induced cardiac fibrosis by inhibiting the activation of Smad3 and MAPK signaling pathways in hVFs. MAPK, mitogen-activated protein kinase; hVFs, human ventricular fibroblasts; ECM, extracellular matrix; p-, phosphorylated; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase; TGF-β1, transforming growth factor-β1.