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

Abstract

BACKGROUND Salvianolic acid B (SalB) is the representative component of phenolic acids derived from the roots and rhizomes of Salvia miltiorrhiza Bge (Labiatae), which has been used widely in Asian countries for clinical therapy of various cardiovascular dysfunction-related diseases. However, cardiac protection effects and the underlying mechanism for clinical application are still poorly understood. Here, we investigated the potential anti-myocardial fibrosis effect and mechanism of SalB on Angiotensin II (Ang II)-induced cardiac fibrosis in vitro. MATERIAL AND METHODS The proliferation and migration capacity of cardiac fibroblasts (CFBs) were measured by MTT assay and scratch analysis, respectively. The colorimetric assay determined the hydroxyproline content in medium. Western blotting detected the protein expressions of nuclear transcription factor-kappa B (NF-κB) pathway-associated proteins, fibronectin (FN), collagen type I (Coll I), α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF). The expression of α-SMA protein was observed by immunofluorescence staining. qRT-PCR detected the mRNA expression of NF-κB. RESULTS SalB attenuated Ang II-induced the proliferation and the migration ability of CFBs. Ang II-induced the extracellular matrix protein Coll I, FN, and α-SMA, the pro-fibrotic cytokine CTGF protein expression was inhibited, and the nuclear translocation of NF-κB p65 subunit was reduced by SalB. Western blotting and qRT-PCR confirmed that SalB blocked the activation of NF-κB induced by Ang II. PDTC (the NF-κB inhibitor) also inhibited proliferation of CFBs and reduced α-SMA and Coll I expression induced by Ang II. CONCLUSIONS SalB can alleviate Ang II-induced cardiac fibrosis via suppressing the NF-κB pathway in vitro.

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.

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.

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.

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.

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.

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.