Salvianolic Acid B-Alleviated Angiotensin II Induces Cardiac Fibrosis by Suppressing NF-κB Pathway In Vitro

Figures

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  Figure 1. Identification of cardiac fibroblasts (CFBs). (A) Morphology image of the second-passage CFBs (magnification, ×100). (B) Representative image of negative control-stained cells (PBS was used instead of primary antibody, magnification, ×200). (C) Representative image of cells stained with the anti-vimentin antibody (magnification, ×200). CFBs – cardiac fibroblasts.

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  Figure 2. Effect of Salvianolic acid B (SalB) on Ang II-induced cardiac fibroblasts (CFBs) proliferation. CFBs were pretreated with SalB (12.5, 25, and 50 μmol/L) or without SalB for 1 h, and then co-incubated with Ang II (1 μmol/L) for 24 h. MTT assay. (i) Control; (ii) Ang II; (iii) SalB (12.5 μmol/L); (iv) SalB (25 μmol/L). (v) SalB (50 μmol/L). Results are presented as the mean ±SEM (* P<0.05 vs. control, ** P<0.01 vs. control; # P< 0.05 vs. Ang II group, ## P<0.01 vs. Ang II group, n=6). Ang II – Angiotensin II; Sal B – salvianolic acid B.

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  Figure 3. Effects of treatments with Salvianolic acid B (SalB) on the migration ability of cardiac fibroblasts (CFBs) induced by Ang II (magnification, ×50). Cells were treated with SalB (12.5, 25, and 50 μmol/L) or without SalB for 1 h, and then co-incubated with Ang II (1 μmol/L) for 24 h. Data are expressed as the mean ±SEM (n=3). * P<0.05 vs. control, ** P<0.01 vs. control; ## P<0.01 vs. Ang II group, ### P<0.001 vs. Ang II group. Ang II – Angiotensin II; Sal B – salvianolic acid B.

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  Figure 4. Effects of Ang II and Salvianolic acid B (SalB) on Ang II-induced upregulation of Coll I, FN, and CTGF in cardiac fibroblasts (CFBs). Cells were treated with Ang II (1 μmol/L) and/or SalB (12.5, 25, and 50 μmol/L, 1 h prior to Ang II stimulation) for 24 h. The expression of Coll I (A), FN (B), and CTGF (C) were identified by Western blot analysis. Data are expressed as mean ±SEM (n=3). * P<0.05 vs. control, ** P<0.01 vs. control; # P<0.05 vs. AngII group, ## P<0.05 vs. AngII group.

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  Figure 5. Salvianolic acid B (SalB) blocks Ang II-induced nuclear transcription factor-kappa B (NF-κB) activation in cardiac fibroblasts (CFBs). Cells were treated with SalB (12.5, 25, and 50 μmol/L) or with SalB (50 μmol/L) for 1 h, and then co-incubated with Ang II (1 μmol/L) for 24 h. Western blotting analysis of (A) p-IκBα, (B) IκBα, (C) p-p65, (D) p65, (E) nuclear factor-κB p65, and (F) cytosolic NF-κB p65. (G) qRT-PCR analysis the mRNA relative expression levels of NF-κB. Data are presented as the mean ± SEM (n=3). ** P<0.01 vs. control; # P<0.05 vs. Ang II group; ## P<0.01 vs. Ang II group. Ang II – Angiotensin II; Sal B – salvianolic acid B; p – phosphorylated; IκBα – inhibitor kappa-Bα; QRT-PCR – quantitative real-time PCR.

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  Figure 6. Salvianolic acid B (SalB) inhibits cardiac fibrosis induced by Ang II via NF-κB signaling pathway in cardiac fibroblasts (CFBs). Cells were pretreated with the inhibitor PDTC (50 μmol/L) for 30 min and SalB (50 μmol/L) for 1 h, and then co-incubated with Ang II (1 μmol/L) for 24 h. (A) MTT assay; (B) Western blot analysis of Coll I expression; (C) Analysis of hydroxyproline (Hyp) content in CFBs supernatant; (D) Immunofluorescence staining of α-SMA in CFBs. α-SMA and nuclei were stained with FITC (green) and DAPI (blue), respectively (magnification, ×200); (E) Western blot analysis of α-SMA expression. Representative Western blot images and quantification are shown. Data are presented as the mean ±SEM (n=3). * P<0.05 vs. control; ** P<0.01 vs. control; # P<0.05 vs. Ang II group; ## P<0.01 vs. Ang II group. Coll I – collagen type I; Ang II – Angiotensin II; SalB – salvianolic acid B; PDTC – pyrrolidine dithiocarbamate; CFBs – cardiac fibroblasts.