Ginsenoside Rb1 Enhances Atherosclerotic Plaque Stability by Improving Autophagy and Lipid Metabolism in Macrophage Foam Cells

Figures

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  FIGURE 1. Rb1 decreased lipid accumulation in macrophage foam cells. Primary peritoneal macrophages from C57BL/6 mice were incubated with ox-LDL (100 μg/ml) for 24 h and further incubated in the absence or presence of different doses of Rb1 for another 24 h. (A,B) oil red O staining was performed to assess lipid accumulation in macrophages.∗p < 0.05, compared to the ox-LDL group, n = 3.

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  FIGURE 2. Rb1 rescued the impaired autophagy flux in macrophage foam cells. (A) Representative western blot analysis of LC3B and SQSTM1/p62. (B) Quantification of LC3 and (C) SQSTM1/p62 expression relative to the β-actin level in different groups. Electron microscopic analysis of autophagosomes in macrophage foam cells treated with Rb1. Primary peritoneal macrophages were incubated with (D) PBS or (E) ox-LDL (100 μg/ml) for 24 h, and further incubated with (F) Rb1 (20 μM ) for another 24 h. Asterisks indicate lipid droplets (LDs); arrows indicate double-membrane vesicles analogous to autophagosomes (APs) in or around LDs. ∗p < 0.05, compared to the ox-LDL group; #p < 0.05, compared to the control group, n = 3.

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  FIGURE 3. Rb1 reduced lipid accumulation of macrophage foam cells by autophagy induction. Primary peritoneal macrophages were transfected with con siRNA or Atg5 siRNA and then treated with ox-LDL (100 μg/ml) in the absence or presence of 20 μM Rb1. (A) Representative western blot analysis of Atg5 and LC3. (B) Quantification of Atg5 and (C) LC3 expression relative to β-actin in macrophages with different treatments. (D) Lipid accumulation in macrophages transfected with Atg5 siRNA with or without Rb1 treatment. (E) Quantitative analysis of lipid accumulation. ∗p < 0.05, compared to con siRNA; #p < 0.05, compared to con siRNA + Rb1, n = 3.

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  FIGURE 4. Effect of Rb1 treatment on plaque stability of apoE-/- mice. (A) Immunohistochemical staining of oil red O staining (lipids), MOMA-2 (macrophages), α-SMA (smooth muscle cells) and sirius red staining for the quantification of collagen. (B) Area measurement of plaque components. Scale bar: 100 μm. ∗p < 0.05, compared to the control group, n = 6.

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  FIGURE 5. Rb1 increased autophagy levels in atherosclerotic plaques in apoE-/- mice. (A) Representative western blot analysis and (B) quantification of LC3-II and SQSTM1/p62 in vivo. ∗p < 0.05, compared to the control group, n = 6.

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  FIGURE 6. Rb1 induced autophagy via promotion of AMPK phosphorylation. (A) Representative western blots and (B) quantification of p-AMPK/AMPK in atherosclerotic plaques of apoE-/- mice treated with Rb1 or saline (control). (C) Representative western blots and (D) quantification of p-AMPK/AMPK in macrophage foam cells treated with Rb1 in the presence or absence of 20 μM compound C (CC). (E) Representative western blots and (F) quantification of LC3-II and SQSTM1/p62 in macrophage foam cells treated with Rb1 in the presence or absence of 20 μM CC. ∗p < 0.05, compared to the ox-LDL group; #p < 0.05, compared to the ox-LDL + Rb1 group; $p < 0.05, compared to the control group, n = 6.

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  FIGURE 7. Schematic diagram of the effects of Rb1 on autophagy. Rb1 increased AMPK phosphorylation and thus induced autophagy activation in macrophage foam cells. Lipid droplets (LDs) are separated by double-membrane vesicles analogous to autophagosomes and delivered to lysosomes to form an autolysosome. LDs are hydrolyzed by lysosomal acid lipase (LAL) into free cholesterols in the autolysosome. Solid lines ( ) depict the mechanism by which Rb1 improves autophagy and lipid metabolism in macrophage foam cells in our present study. Dotted lines( ) represent a possible signaling molecule (mTOR) that may serve as an upstream regulator of autophagy induced by Rb1, but was not investigated in our study.