Experimental Validation of Antiobesogenic and Osteoprotective Efficacy of Ginsenoside CK via Targeting Lipid and Atherosclerosis Pathways

Figures

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  Figure 1. The MTT assay was used to measure cell viability. CK (3.125–50 μM) was administered to (A) 3T3-L1 pre-adipocyte (Orange-colored columns) and (B) MC3T3-E1 pre-osteoblast cells (1 × 104 cells/well) (Tan-colored columns) for 24 h. A two-tailed Student’s t-test was used to determine whether there was a substantial distinction between the groups; ns denotes a non-significant change, * p < 0.05, ** p < 0.01 when compared to a control group (Black-colored column) that was not treated.

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  Figure 2. The inhibitory effect of CK on lipid accumulation in MDI-induced 3T3-L1 adipocytes was assessed as follows: (A) Oil Red O staining was performed to visualize fat droplets, which were then observed under a light microscope at 20× magnification. (B) Lipid accumulation was quantified by measuring the absorbance of Oil Red O dissolved in isopropyl alcohol at 520 nm. Data represent the mean ± SEM from three independent experiments. Statistical significance is indicated as ### p < 0.05, ** p < 0.01, *** p < 0.001, compared to the MDI-treated group.

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  Figure 3. (A) The proportion of a-glucosidase inhibition at various CK concentrations. Acarbose, or ACR, was employed as a control. We also analyzed the half-maximum inhibitory concentration (IC50) of CK and ACR (B) 3T3L1 cells using the 2-NBDG uptake test. Using a fluorescent derivative of glucose 2-NBDG, the impact of CK on 3T3L1 cells’ glucose absorption was examined for 24 h with and without CK. Insulin was employed as a positive control at 100 nM. Data are presented as a control percentage. *** p < 0.001.

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  Figure 4. The relative expression of inflammatory genes (TNFα, IL-6), adipogenic genes (PPARγ, C/EBPα, LPL), the lipogenic gene FAS, and the thermogenic gene UCP1, along with TP53, was measured in differentiated 3T3-L1 cells treated with CK at concentrations of 2.5 μM and 10 μM, or RSG at 20 μM. Results are presented as the mean ± standard deviation from three independent experiments. Statistical significance was assessed using a two-tailed Student’s t-test. Significant differences in gene expression between untreated and treated groups are indicated as ** p < 0.01, and *** p < 0.001.

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  Figure 5. (A) ALP activity in CK and E2-treated differentiated MC3T3-E1 cells. (B) The impact of CK on MC3T3-E1 cell mineralization. A 96-well plate was used to measure the quantity of Alizarin Red S at 562 nm. (C) The calcium-binding Alizarin Red S dye was used to evaluate the calcium deposits in the extracellular matrix for matrix mineralization. Up to Day 28, CK treatment accelerated the mineralization of the extracellular matrix. The images are representative of over three different concentrations of CK and E2. The results are presented as the mean ± standard deviation from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test. Significant differences in lipid production compared to the untreated (control) group are denoted by ** p < 0.01, and *** p < 0.001.

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  Figure 6. Collagen content in MC3T3-E1 cells is increased by CK extracts. For 12 days, cells were exposed to extracts at doses ranging from 2.5 to 10 μM, either with or without a differentiation medium. E2 was used at a concentration of 100 nm. (A) Picro-Sirius red staining was carried out and seen using a microscope (magnification of ×100). (B) Absorbance was measured at 550 nm to determine the amount of collagen. The presented data are the mean ± standard deviation (SD) of three studies. Statistical analysis revealed significant differences, denoted as * p < 0.1, *** p < 0.001 when compared with the indicated ascorbic acid and β-glycerophosphate treated group.

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  Figure 7. Relative expression of proinflammatory cytokines TNFα, and IL-6, and osteogenic genes RunX2, ALP, BGLAP, OCN, and COL1a1 with TP53 in differentiated MC3T3-E1 cells on treatment with CK at 2.5 μM and E2 at 100 nm concentrations, respectively. A two-tailed Student’s t-test was used to determine whether the difference was statistically significant. The non-treated and treated groups’ significant differences in gene expression are indicated by * p < 0.1, ** p < 0.01, *** p < 0.001 vs. control.