Ginsenoside Rb1 Ameliorates Diabetic Arterial Stiffening via AMPK Pathway

Figures

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  FIGURE 1. Rb1 improves aortic compliance and restores acetylcholine-induced endothelium-dependent vasorelaxation. (A) Chemical structure of ginsenoside Rb1. (B) After the addition of phenylephrine, cumulative doses of acetylcholine (1 × 10–9–1 × 10–5.5 M) were added to check the endothelial functions. (C) Peterson’s elastic modulus (Ep). (D) Arterial stiffness index (β). (E) Cross-sectional compliance (CSC), (F) cross-sectional distensibility (CSD), (G) compliance coefficient (CC), and (H) distensibility coefficient (DC). Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM.

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  FIGURE 2. Rb1 reduces DM-induced aortic remodeling (A) Sirius red staining (bright field and dark field) (scale bar 50 μm). Collagen fibers were stained bright red. Semiquantitative analysis of collagen fibers. Perivascular collagen content, shown as the perivascular collagen area/luminal area (PVCA/LA) ratio. (B) Elastic fibers shown by Verhoeff-Van Gieson staining (scale bar 50 μm). Elastic fibers are black, VSMCs are light red, and collagen fibers are pink. Semiquantitative analysis of elastic fibers. (C) Fibronectin accumulation (green: fibronectin, red: elastic lamina, blue: nuclei, scale bar 50 μm). Semiquantitative analysis of fibronectin expression. Representative immunohistochemical staining (D), Western blot bands (F), and semiquantitative analysis of collagen I expression (scale bar 20 μm). Representative immunohistochemical staining (E), Western blot bands (G), and semiquantitative analysis of collagen Ⅲ expression (scale bar 20 μm). Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM.

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  FIGURE 3. AMPK pathway is involved in the effect of Rb1 on DM- and HG-mediated TGFβ1, Smad2/3, and collagen expression. Representative Western blot bands and semiquantitative analysis of (A) phosphorylated AMPK (p-AMPK), (B) TGFβ1, (C) phosphorylated Smad2 (p-Smad2), and (D) phosphorylated Smad3 (p-Smad3) in aorta extracts. (E–G) VSMCs were treated with Rb1 at doses of 10, 20, 40, and 60 μM in a high-glucose medium. Representative Western blot bands and semiquantitative analysis of phosphorylated AMPK (p-AMPK), TGFβ1, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3). Semiquantitative analysis of above proteins expressions. Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM and HG + Rb1 vs. HG.

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  FIGURE 4. Rb1-mediated reduction of TGFβ1 and phospho-Smad2/3 and collagen accumulation are partly abolished by treatment with compound C, an inhibitor of AMPK. VSMCs were treated with Rb1 at doses of 40 μM in a high-glucose medium and pretreated with compound C. Representative Western blot bands and semiquantitative analysis of (A) phosphorylated AMPK (p-AMPK), (B) TGFβ1, and (C) phosphorylated Smad2 (p-Smad2). (D) Phosphorylated Smad3 (p-Smad3). (E) Collagen I and (F) collagen III. Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 HG + Rb1 vs. HG.

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  FIGURE 5. Rb1 suppresses MMP-2 and MMP-9 expression. Representative Western blot bands and semiquantitative analysis of (A) MMP-2 and (B) MMP-9 protein expression in aorta. Representative Western blot bands and semiquantitative analysis of (C) MMP-2 and (D) MMP-9 protein expression in VSMCs. Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM and HG + Rb1 vs. HG.

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  FIGURE 6. Rb1 improves DM and HG-induced oxidative stress. (A) Immunohistochemical staining and semiquantitative analysis of 3-nitrotyrosine (3-NT). ROS was assessed by three different methods: (B) DCFH-DA, (C) Amplex Red, and (F) DHE in VSMCs (D) ROS levels. in each group. Original magnification × 200. (E) Semiquantitative analysis of ROS levels in each group. The positive control is the active oxygen donor from the DCFH-DA kit, which contains H2O2. Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM and HG + Rb1 vs. HG.

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  FIGURE 7. NOX isoforms involved in the effects of Rb1 on DM-induced oxidative stress. Quantitative analysis of (A) NOX1 (B) NOX2 and (C) NOX4 mRNA expression in the aorta. Quantitative analysis of (D) NOX1 (E) NOX2 and (F) NOX4 mRNA expression in VSMCs. Representative Western blot bands and semiquantitative analysis of (H) NOX1 (I) NOX2 and (G) NOX4 protein expression in VSMCs. Data are mean ± SEM. n = 5–6, *p < 0.05 vs. Control; #p < 0.05 DM + Rb1 vs. DM and HG + Rb1 vs. HG.