Ginsenoside Rb1 Prevents Dysfunction of Endothelial Cells by Suppressing Inflammatory Response and Apoptosis in the High-Fat Diet Plus Balloon Catheter-Injured Rabbit Model via the G Protein-Coupled Estrogen Receptor-Mediated Phosphatidylinositol 3-Kinases (PI3K)/Akt Pathway

Abstract

<strong>BACKGROUND</strong> The initiation of atherosclerosis (AS) is attributed to the dysfunction of endothelial cells (ECs) via the inhibition of g protein-coupled estrogen receptor (GPER). In the current study, we assessed the potential of Ginsenoside Rb1 (Rb1) to attenuate the dysfunction of ECs via GPER-mediated PI3K/Akt pathway. <strong>MATERIAL AND METHODS</strong> AS was induced in rabbits and then the AS rabbits were treated with Rb1. Thereafter, the ECs were isolated from AS and healthy rabbits, and treated with Rb1. The effect of Rb1 on blood lipid levels in AS rabbits and on apoptosis, inflammatory response, and GPER/PI3K/Akt axis activity in ECs was detected. Furthermore, the activities of GPER and PI3K were modulated to verify the key role of the axis in the anti-AS effect of Rb1. <strong>RESULTS</strong> The levels of total cholesterol, low-density lipoprotein (LDL), and triglyceride in AS rabbits were suppressed by Rb1 while the high-density lipoprotein (HDL) level was increased. In in vitro assays, Rb1 administration inhibited apoptosis process and the production of pro-inflammation cytokines in AS ECs. The expression levels of GPER, p-PI3K, and p-Akt were upregulated by Rb1, associated with the increased level of Bcl-2 and reduced level of Bax. When the activity of GPER was inhibited by GP-15 in AS ECs, the treatment effect of Rb1 was blocked. However, the activation of PI3K could restore the protective effect of Rb1 after the inhibition of GPER. <strong>CONCLUSIONS</strong> The anti-AS potential of Rb1 was exerted by restoring the regular function of ECs via the activation of GPER-mediated PI3K/Akt signaling.

Figure 1

Administration of Rb1 attenuated lipid metabolism disorder induced by high-fat diet plus balloon catheter-injured model. Rabbits were subjected to high-fat diet plus balloon catheter-injured surgery and treated with Rb1 of 5, 10, and 20 mg/kg body weight. (A) Quantitative analysis results of ELISA detection of blood total cholesterol. (B) Quantitative analysis results of ELISA detection of blood HDL. (C) Quantitative analysis results of ELISA detection of blood LDL. (D) Quantitative analysis results of ELISA detection of blood triglyceride. * P<0.05 vs. Control group. ^# P<0.05 vs. HCD group. Each assay was performed 5 times.

Figure 2

Administration of Rb1 inhibited inflammatory response and apoptosis in dysfunctional ECs. ECs were isolated from AS and health rabbits and administrated with Rb1 of 20, 40, and 80 μM. (A) quantitative analysis results of ELISA detection of blood IL-6. (B) Quantitative analysis results of ELISA detection of blood IL-1β. (C) Quantitative analysis results of ELISA detection of blood TNF-α. (D) Representative images and quantitative analysis of flow cytometry detection of apoptosis. * P<0.05 vs. Control group. ^# P<0.05 vs. HCD group. Each assay was performed 5 times.

Figure 3

Administration of Rb1 activated GPER/PI3K/Akt axis dysfunctioned in ECs. ECs were isolated from AS and health rabbits and administrated with Rb1 of 20, 40, and 80 μM for 24 h. (A) Representative images and quantitative analysis of western blotting detection of molecule expressions in GPER/PI3K/Akt axis. (B) Representative images of immunofluorescence detection of GPER expression and distribution. Magnification, 400×. * P<0.05 vs. Control group. ^# P<0.05 vs. HCD group. Each assay was performed 5 times.

Figure 4

Anti-AS function of Rb1 depended on the activation GPER- mediated PI3K/Akt pathway. ECs were isolated from AS and health rabbits and administrated with 80 μM Rb1, 100 nM G-15, and 25 mg/mL 740 Y-P for 24 h. (A) Representative images and quantitative analysis of western blotting detection of molecule expressions in GPER/PI3K/Akt axis. (B) Representative images and quantitative analysis of flow cytometry detection of apoptosis. (C) Quantitative analysis results of ELISA detection of blood IL-6. (D) Quantitative analysis results of ELISA detection of blood IL-1β. (E) Quantitative analysis results of ELISA detection of blood TNF-α. * P<0.05 vs. HCD+H group. ^# P<0.05 vs. HCD+H+G-15 group. Each assay was performed 5 times.