Ginsenoside F1 Ameliorates Endothelial Cell Inflammatory Injury and Prevents Atherosclerosis in Mice through A20-Mediated Suppression of NF-kB Signaling

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  FIGURE 1. Ginsenoside F1 reduced atherosclerotic plaque development in ApoE-/- mice. All mice were fed HFD. ApoE-/- mice were treated with GF1 (50 mg/kg/day, i.g.), probucol (2 mg/kg, i.g.), or its vehicle (as model group) for 8 weeks; C57 mice were used as the control group. (A) Representative light photomicrographs of oil red O-stained sections from the aortic root. (B) Statistical analysis of the oil red O-stained sections. (C) Representative images of Sudan IV-stained en face of the proximal aorta. (D) Statistical analysis of aortic AS expressed as a fraction of the total aortic area. Values (n = 10 per group) are expressed as the mean ± SD. Values (n = 10 per group) are expressed as the mean ± SD. ##p < 0.01, vs. C57 control group; ∗p < 0.05, ∗∗p < 0.01 vs. the vehicle-treated ApoE-/- model group.

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  FIGURE 2. Ginsenoside F1 ameliorated inflammatory responses in HFD-induced ApoE-/- mice. All mice were fed HFD. ApoE-/- mice were treated with GF1 (50 mg/kg/day, i.g.), probucol (2 mg/kg/day, i.g.), or its vehicle (as model group) for 8 weeks; C57 mice were used as the control group. (A) Representative photomicrographs of MPO distribution in the whole body. (B) Statistical analysis of MPO-positive average luminescence intensity. Values (n = 10 per group) are expressed as the mean ± SD. ##p < 0.01, vs. C57 control group; ∗p < 0.05 vs. the vehicle-treated ApoE-/- model group.

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  FIGURE 3. Ginsenoside F1 decreased LOX-1 and TLR4 expression at the aortic valve in ApoE-/- mice. All mice were fed HFD. ApoE-/- mice were treated with GF1 (50 mg/kg/day, i.g.), probucol (2 mg/kg/day, i.g.), or its vehicle (as model group) for 8 weeks; C57 mice were used as the control group. (A) Representative images of LOX-1 expression under immunofluorescent staining. (B) Quantitative analysis of LOX-1 expression in aortic valve. (C) Representative images of TLR4 expression under immunofluorescent staining. (D) Quantitative analysis of TLR4 expression in aortic valve. (E) Representative images of NF-κB expression under immunofluorescent staining. (F) Quantitative analysis of NF-κB expression in aortic valve. Values (n = 10 per group) are expressed as the mean ± SD. ##p < 0.01, vs. C57 control group; ∗p < 0.05 vs. the vehicle-treated ApoE-/- model group.

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  FIGURE 4. Ginsenoside F1 prevented ox-LDL-induced HUVECs inflammatory injury. (A) Effects of different concentration and time of ox-LDL on HUVECs viability. (B) Effects of GF1 on ox-LDL-induced HUVECs viability. HUVECs were pretreated with the indicated doses (1, 4, and 16 μM) of GF1 for 24 h before exposure to 70 μg/ml ox-LDL for another 24 h. Cell viability was measured by the MTT assay. (C) Effects of GF1 on the viability of HUVECs. Cells were incubated with the indicated doses (1, 4, and 16 μM) of GF1 for 24 h. Cell viability was measured by the MTT assay. (D) HUVECs were pretreated with GF1 (16 μM) for 24 h and ox-LDL incubation for 24 h. Proteins of the supernatant related to the inflammatory factors were analyzed using human inflammation antibody array. All of the results are expressed as the mean ± SD of three independent experiments. ##p < 0.01, vs. control group; ∗p < 0.05, ∗∗p < 0.01 vs. ox-LDL group, NG means no significance.

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  FIGURE 5. Ginsenoside F1 mitigated the adhesion of THP-1 cells to HUVECs stimulated by ox-LDL. HUVECs were pre-treated with GF1 (16 μM), followed by treatment with ox-LDL for another 24 h. (A) Representative images of CM-diL labeled THP-1 cells adherent to HUVECs. (B) The quantitative analysis of relative fluorescence of CM-diL labeled THP-1 cells adherent to HUVECs. All of the results are expressed as the mean ± SD of three independent experiments. ##p < 0.01, vs. control group; ∗p < 0.05, ∗∗p < 0.01 vs. ox-LDL group.

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  FIGURE 6. Ginsenoside F1 suppressed LOX-1, TLR4, and blocked NF-κB nuclear translocation in ox-LDL-treated HUVECs. HUVECs were pre-treated with various concentrations of GF1, followed by treatment with ox-LDL for another 24 h. (A) Protein expression levels of LOX-1 and TLR4 were measured via western blotting. (B) Densitometric analysis was used to quantify the levels of LOX-1 and TLR4. (C) Gene expression levels of LOX-1 and TLR4 were measured via real-time PCR. (D,E) Protein expression levels of p-IκB, IκB, cytoplasm NF-κB, nucleus NF-Kb, total NF-κB, β-actin and Lamin B were measured via western blotting. (F) Representative image of NF-κB activation was evaluated using electrophoretic mobility shift assay. (G) Densitometric analysis of NF-κB activation. (H) Densitometric analysis was used to quantify the levels of p-IκB, IκB, cytoplasm NF-κB, nucleus NF-κB, and total NF-κB. All of the results are expressed as the mean ± SD of three independent experiments. ##p < 0.01, vs. control group; ∗p < 0.05, ∗∗p < 0.01 vs. ox-LDL group.

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  FIGURE 7. Ginsenoside F1 inhibited NF-κB signaling via A20 promotion. (A) HUVECs were pre-treated with various concentrations of GF1, followed by treatment with ox-LDL for another 24 h. Protein expression levels of A20 and β-actin were measured via western blotting. (B) Densitometric analysis was used to quantify the levels of A20. (C) A20 was knocked down by siRNA, as described in Materials and Methods. At 24 h post-transfection, cells were incubated with GF1 (16 μM) for 12 h and then exposed to ox-LDL for another 24 h. Protein expression levels of A20, IL-6, ICAM-1, cytoplasm NF-κB, nucleus NF-κB and β-actin were measured via western blotting. (D) Densitometric analysis was used to quantify the levels of A20, IL-6, ICAM-1, cytoplasm NF-κB, and nucleus NF-κB. (E) Docking simulation of GF1 into A20. All of the results are expressed as the mean ± SD of three independent experiments. ##p < 0.01, vs. control group; ∗p < 0.05, ∗∗p < 0.01 vs. ox-LDL group; p$$< 0.01 vs. ox-LDL+GF1 group.

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  FIGURE 8. Schematic of GF1 mechanism of preventing ox-LDL-induced inflammatory injuries through activating A20-mediated NF-κB signal pathway.