Therapeutic Effects of Salvianolic Acid B on Angiotensin II–Induced Atrial Fibrosis by Regulating Atrium Metabolism via Targeting AMPK/FoxO1/miR-148a-3p Axis

Fig. 1. Sal B attenuated Ang II–induced atrial fibrosis. A Masson’s trichrome staining of hearts from sham and Ang II–treated mice and Ang II + Sal B–treated mice. Scale bar = 100 μm and 20 μm. B Quantification of interstitial fibrosis by calculating collagen volume fraction 4 weeks after operation. C HE staining of hearts from sham and Ang II–treated mice and Ang II + Sal B–treated mice. Scale bar = 100 μm and 20 μm. D, E Western blot analysis showed the protein expression levels of α-SMA, Col1a1, and CTGF in atria tissues from sham and Ang II–treated mice and Ang II + Sal B–treated mice. F, G Immunofluorescence staining analysis showed the protein expression levels of Col3a1 in atria tissues from sham and Ang II–treated mice and Ang II + Sal B–treated mice. Scale bar = 50 μm. Data were presented as mean ± SEM (n = 8 in each group). *p < 0.05 vs Sham group; #p < 0.05 vs Ang II group

Fig. 2. Effects of Sal B on the proliferation, secretion functions, and oxidative stress of MFBs. A Untreated FBs (control), Ang II–treated MFBs, and Ang II + Sal B–treated MFBs were stained with α-SMA (green) and DAPI (blue). Scale bar = 20 μm. B The fluorescence signals of different groups were analyzed using ImageJ. C, D Representative fluorescence images of EdU staining of FBs. Proliferating FBs positively stained with EdU showed red color. Cell nuclei stained with DAPI showed blue color. Scale bar = 100 μm. E Cell viability treated with PBS (control), Ang II, or dedicated concentrations of Sal B (12.5 μg/ml, 25 μg/ml, or 50 μg/ml). F ELISA assay showed the level of GSH in the control group, Ang II group, and Ang II + Sal B group. G ROS level was examined in the control group, Ang II group, and Ang II + Sal B group. Data were presented as mean ± SEM (n = 3 in each group). *p < 0.05 vs control group, **p < 0.01 vs control group; #p < 0.05 vs Ang II group, ##p < 0.01 vs Ang II group; @p < 0.05, @@p < 0.01; ns, no significance

Fig. 3. Multivariate statistical analysis of the cell metabolites in Ang II–activated FBs. A Distribution of intensities for metabolites in control, Ang II, and Ang II + Sal B. B Principal component analysis (PCA) score plot of the control (blue squares), Ang II (red triangles), and Ang II + Sal B (yellow rhombus) groups. C PLS-DA score plot of the control (blue squares), Ang II (red triangles), and Ang II + Sal B (yellow rhombus) groups. D OPLS-DA models of control and Ang II. The blue squares and red triangles indicated control and Ang II groups, respectively. E OPLS-DA models of Ang II + Sal B and Ang II. The red triangles and yellow rhombus indicated Ang II and Ang II + Sal B groups, respectively. F Cross-validated score plots of the OPLSDA model (R2 = 0.816; Q.2 = 0.972)

Fig. 4. Metabonomics pathway analysis. Based on the impact value > 0.05, two perturbed metabolic pathways, including purine metabolism and FoxO signaling pathway were significantly affected by Sal B

Fig. 5. Sal B induced AMPK/FoxO1 activation and miR-148a-3p expression in Ang II–activated MFBs. A Western blot showed that the protein expressions of α-SMA, Col1a1, and CTGF in control, Ang II, Ang II + Sal B, and Sal B + Compound C (AMPK antagonist) groups. B Western blot showed that the phosphorylation of AMPK and FoxO1 in each group. C RT-PCR analysis of AMPK and FoxO1 levels in each group. Data were normalized to GAPDH. D The levels of miR-148a-3p in untreated FBs (control), Ang II–treated FBs, and Ang II + Sal B–treated FBs and Sal B + Compound C–treated FBs after 24 h. Data were normalized to U6. Data were presented as mean ± SEM (n = 3 in each group). *p < 0.05 vs control group, **p < 0.01 vs control group; #p < 0.05 vs Ang II group, ##p < 0.01 vs Ang II group; $p < 0.05 vs Ang II + Sal B group, $$p < 0.01 vs Sal B + Compound C group

Fig. 6. Schematic diagram of the perturbed metabolic pathways regulated by Sal B for the beneficial effects in Ang II–activated cardiac fibroblasts. Green and red represent reduction and increase in the Sal B–treated group compared with the Ang II group, respectively