Anti-Thrombotic and Metabolic Protective Effects of Ginseng in High-Fat Diet-Induced Obese Rats: In Vivo Evaluation with In Silico Mechanistic Prediction

Figures

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  Figure 1. Schematic diagram of the experimental protocol. Rats were acclimated for 1 week and fed a high-fat diet (HFD, 60% fat diet) for 4 weeks (pre-experimental period) to induce an animal model of obesity. The animals on the HFD for 4 weeks were weighed and randomly assigned to five groups (placebo (HFD), KGF 50 mg/kg (KGF 50), KGF 100 mg/kg (KGF 100), KGF 200 mg/kg (KGF 200), and aspirin (ASP)) so that the mean body weight was approximately equal between groups. The control group (CONT) was fed a standard chow diet until the end of the experiment. The animals were sacrificed after 6 weeks.

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  Figure 2. HPLC-UV chromatograms (203 nm) of standard overlay (A) and KGF (B). HPLC-UV, high-performance liquid chromatography with ultraviolet detection; KGF, KoreaGinseng F Max.

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  Figure 3. Effect of KGF administration on body weight, food consumption, and weight of the liver and epididymal adipose tissue. (A) Changes in mean Body weight of the experimental animals fed a standard chow diet (n = 9) and an HFD (n = 45) during the Pre-experimental period. (B) The average Changes in Body weight of the six experimental groups during the Experimental period. (C) Net weight changes and (D) daily Food consumption changes over the 6-week Experimental period. (E) Average weight of Liver and (F) epididymal adipose tissue of experimental animals at the end point of the experiment. All values were expressed as mean ± S.D. p-values were determined by one-way ANOVA. * p < 0.05 and ** p < 0.01; significant as compared with CONT group.

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  Figure 4. Effect of KGF administration on changes in serum lipid levels in the HFD-induced obese animal model. (A) Total cholesterol, (B) Triglyceride, (C) high-density lipoprotein (HDL), and (D) low-density lipoprotein (LDL) levels in whole blood were measured in the six experimental animal groups. All values were expressed as mean ± S.D. of seven rats. p-values were determined by one-way ANOVA. * p < 0.05 and ** p < 0.01, significant as compared with CONT group; # p < 0.05 and ## p < 0.01, significant as compared with HFD group.

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  Figure 5. Effect of KGF administration on changes in serum (A) angiotensin-converting enzyme (ACE), (B) adiponectin, and (C) leptin levels in the HFD-induced obese animal model. All values were expressed as mean ± S.D. of seven rats. p-values were determined by one-way ANOVA. *** p < 0.001, significant as compared with CONT group; # p < 0.05 and ## p < 0.01, significant as compared with HFD group.

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  Figure 6. Effect of KGF administration on changes in serum (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) blood urea nitrogen (BUN), and (D) creatinine levels in the HFD-induced obese animal model. There were no statistical differences between the six groups for any of the measurements. All values were expressed as mean ± S.D. of seven rats. p-values were determined by one-way ANOVA.

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  Figure 7. Effect of KGF administration on changes in adipocyte size. (A) Representative H&E-stained images of periepididymal adipocytes. (B) Mean diameter and (C) mean area of adipocytes in six experimental groups. Scale bars in (A) equal 100 μm. All values are expressed as mean ± S.D. of seven rats. p-values were determined by one-way ANOVA. **** p < 0.0001, significant as compared with CONT group; ## p < 0.01, ### p < 0.001, and #### p < 0.0001, significant as compared with HFD group.

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  Figure 8. Effect of KGF administration on changes in hepatic steatosis. (A) Representative histological images of hepatocytes and (B) histomorphometric analysis of the steatosis area. The arrows in (A) indicate the area of steatosis. Scale bars in (A) equal 100 μm. All values are expressed as mean ± S.D. of seven rats. p-values were determined by one-way ANOVA. **** p < 0.0001, significant as compared with CONT group; ### p < 0.001, significant as compared with HFD group.

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  Figure 9. Time−course changes in arterial blood flow after FeCl3 application wrapped around the carotid artery using a real-time microcirculation imaging system. (A) Dissection image of a rat with a midline incision in the neck and an open common carotid artery (CCA). (B) Microscopic image of the CCA (dotted square) where the blood flow rate is measured, and (C) its Doppler blood flow image. (D) Graph showing the arterial perfusion unit before and after exposure to FeCl3. Note the significant decline in perfusion unit with FeCl3 exposure (arrow). (E) Graph of blood flow rates by group before and after FeCl3 exposure converted to percentage values. Mean blood flow units in the 10 min prior to FeCl3 exposure were standardized to 100%. (F) Serial Doppler blood flow images from each experimental group taken at 5 min intervals (5, 10, 15, and 20 min) after exposure to FeCl3. The carotid artery is red during the states at high blood flow, green at moderate blood flow, and blue at low blood flow.

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  Figure 10. Network pharmacology analysis of white-ginseng-derived ginsenosides in anti-thrombotic-related egenes. (A) Chemical structures of the three major ginsenosides: Rg1, Rb1, and Rf. (B) Venn diagram showing the overlap between predicted targets of the three ginsenosides and anti-thrombotic-related genes. (C) GO biological process enrichment analysis of overlapping target genes, with the top 20 enriched terms visualized as a bubble plot. (D) KEGG pathway enrichment analysis of shared target genes associated with anti-thrombotic-related genes.

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  Figure 11. Network pharmacology analysis of white-ginseng-derived ginsenosides related to anti-thrombotic activity: GO–key gene–KEGG network of the eight shared genes. The network was constructed using Cytoscape based on the GO and KEGG enrichment results from Metascape. The central red nodes represent the eight shared genes (STAT3, PTAFR, VEGFA, FGF2, HPSE, IL2, HSP90AA1, and LGALS3). The left side shows enriched GO biological process terms, and the right side shows enriched KEGG pathways. Red KEGG pathway nodes indicate the representative predicted pathways prioritized in this study, including the PI3K–Akt signaling pathway, Th17 cell differentiation, proteoglycan/ECM-related signaling, and the calcium signaling pathway. Light purple KEGG nodes indicate other enriched pathways. Edges indicate gene–term or gene–pathway associations.

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  Figure 12. Molecular docking analysis of marker ginsenosides against the eight overlapping target proteins (STAT3, PTAFR, VEGFA, FGF2, HPSE, IL2, HSP90AA1, and LGALS3). (A) Heatmap showing the docking affinity values between each single compound and target protein. Stronger predicted binding affinity is indicated in red. (B) Representative docking poses of the two highest affinity interactions selected for each single compound. Values marked as 0* represent unsuccessful or energetically unfavorable docking results and were not interpreted as stable binding. Abbreviations: STAT3, signal transducer and activator of transcription 3; PTAFR, platelet-activating factor receptor; VEGFA, vascular endothelial growth factor A; FGF2, fibroblast growth factor 2; HPSE, heparanase; IL2, interleukin-2; HSP90AA1, heat shock protein 90 alpha family class A member 1; LGALS3, galectin-3.