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. 2018 Jul 24:2018:6712585.
doi: 10.1155/2018/6712585. eCollection 2018.

Sodium Tanshinone IIA Sulfonate Prevents Angiotensin II-Induced Differentiation of Human Atrial Fibroblasts into Myofibroblasts

Tangting Chen  1 , Miaoling Li  1 , Xuehui Fan  1 , Jun Cheng  1 , Liqun Wang  2
Affiliations

Sodium Tanshinone IIA Sulfonate Prevents Angiotensin II-Induced Differentiation of Human Atrial Fibroblasts into Myofibroblasts

Tangting Chen et al. Oxid Med Cell Longev. .

Abstract

Differentiation of atrial fibroblasts into myofibroblasts plays a critical role in atrial fibrosis. Sodium tanshinone IIA sulfonate (DS-201), a water-soluble derivative of tanshinone IIA, has been shown to have potent antifibrotic properties. However, the protective effects of DS-201 on angiotensin II- (Ang II-) induced differentiation of atrial fibroblasts into myofibroblasts remain to be elucidated. In this study, human atrial fibroblasts were stimulated with Ang II in the presence or absence of DS-201. Then, α-smooth muscle actin (α-SMA), collagen I, and collagen III expression and reactive oxygen species (ROS) generation were measured. The expression of transforming growth factor-β1 (TGF-β1) and the downstream signaling of TGF-β1, such as phosphorylation of Smad2/3, were also determined. The results demonstrated that DS-201 significantly prevented Ang II-induced human atrial fibroblast migration and decreased Ang II-induced α-SMA, collagen I, and collagen III expression. Furthermore, increased production of ROS and expression of TGF-β1 stimulated by Ang II were also significantly inhibited by DS-201. Consistent with these results, DS-201 significantly inhibited Ang II-evoked Smad2/3 phosphorylation and periostin expression. These results and the experiments involving N-acetyl cysteine (antioxidant) and an anti-TGF-β1 antibody suggest that DS-201 prevent Ang II-induced differentiation of atrial fibroblasts to myofibroblasts, at least in part, through suppressing oxidative stress and inhibiting the activation of TGF-β1 signaling pathway. All of these data indicate the potential utility of DS-201 for the treatment of cardiac fibrosis.

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Figures

Figure 1
Figure 1
The molecular structure of DS-201.
Figure 2
Figure 2
Effects of DS-201 on cell viability. Human atrial fibroblasts were exposed to DS-201 (0, 5, 25, 50,100, and 200 μM) for 24 h. Cell viability was measured using the CCK8 assay. Data shown are mean ± SD of 4 independent experiments, presented as % of the control value (first bar).
Figure 3
Figure 3
DS-201 prevents Ang II-induced fibrotic response in atrial fibroblasts. Atrial fibroblasts were exposed to Ang II (0.5 μM) with or without DS-201 (0, 5, 25, 50, and 100 μM) for 24 h. (a) Expression of α-SMA was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of α-SMA normalized to GAPDH was calculated. (b) Expression of collagen I was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of collagen I normalized to GAPDH was calculated. (c) Expression of collagen III was also analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of collagen III normalized to GAPDH was calculated. All data shown are mean values ± SD and are expressed as fold changes. ∗∗ P < 0.01 versus control (first bar); # P < 0.05 versus Ang II; ## P < 0.01 versus Ang II.
Figure 4
Figure 4
DS-201 prevents Ang II-induced cell migration and cell proliferation. (a) Atrial fibroblasts were added to upper chambers containing porous filters and then treated with Ang II (0.5 μM) with or without DS-201 (0, 5, 25, 50, and 100 μM). After 24 h, the cells were fixed and were stained with crystal violet. The cells that had migrated into the lower chambers were counted. Representative images of cell migration are shown (scale bar, 200 μm). (b) Quantitative assessment of 3 independent cell migration experiments was performed. (c) Atrial fibroblasts were stimulated with Ang II in the absence or in the presence of DS-201 (5, 25, 50, and 100 μM) for 24 h. Then, cell proliferation was measured by CCK8 assay. All data shown are mean values ± SD and are expressed as % of the control value (first bar). ∗∗ P < 0.01 versus control (first bar); ## P < 0.01 versus Ang II.
Figure 5
Figure 5
DS-201 inhibits Ang II-induced oxidative stress. Atrial fibroblasts were exposed to Ang II (0.5 μM) with or without DS-201 (0, 5, 25, 50, and 100 μM) for 1 h. (a) Cells were stained with DCFH-DA and the fluorescence intensity of DCF was measured at 488/525 nm using a microplate reader. (b) The DCF fluorescence was also monitored by fluorescence microscopy, and representative images of 3 independent experiments are shown (scale bar, 100 μm). (c, d) The level of SOD and CAT was measured by the respective kits according to the manufacturer's instructions. All data shown are mean ± SD for 3 independent experiments and presented as % of the control value (first bar). ∗∗ P < 0.01 versus Con; # P < 0.05 versus Ang II; ## P < 0.01 versus Ang II.
Figure 6
Figure 6
NAC inhibits Ang II-induced fibrotic response in atrial fibroblasts. Atrial fibroblasts were pretreated with NAC (10 mM) for 1 h, followed by stimulation with Ang II (0.5 μM) for 24 h. Expression of α-SMA, collagen I, and collagen III was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of α-SMA, collagen I, and collagen III normalized to GAPDH was calculated. All data shown are mean values ± SD and are expressed as fold changes. ∗∗ P < 0.01 versus control (first bar); ## P < 0.01 versus Ang II.
Figure 7
Figure 7
DS-201 prevents Ang II-induced TGF-β1 activation in human atrial fibroblasts. Atrial fibroblasts were exposed to Ang II (0.5 μM) with or without DS-201 (0, 5, 25, 50, and 100 μM) for 24 h. (a) Expression of TGF-β1 was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of TGF-β1 normalized to GAPDH was calculated. (b) Phosphorylation (p) of Smad2/3 was analyzed by western blotting. Representative images of 3 independent experiments and densitometric analysis of phosphorylated Smad2/3 normalized to total Smad2/3 are shown. (c) Expression of periostin was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ration of periostin normalized to GAPDH was calculated. ∗∗ P < 0.01 versus control (first bar); ## P < 0.01 versus Ang II.
Figure 8
Figure 8
The blockade of TGF-β1 inhibits Ang II-induced fibrotic response in atrial fibroblasts. Atrial fibroblasts were pretreated with an anti-TGF-β1 antibody (2 μg/mL) for 1 h, followed by stimulation with Ang II (0.5 μM) for 24 h. Expression of α-SMA, collagen I, and collagen III was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of α-SMA, collagen I, and collagen III normalized to GAPDH was calculated. All data shown are mean values ± SD and are expressed as fold changes. ∗∗ P < 0.01 versus control (first bar); ## P < 0.01 versus Ang II.
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