Ginsenoside Rg3-enriched Korean Red Ginseng extract attenuates Non-Alcoholic Fatty Liver Disease by way of suppressed VCAM-1 expression in liver sinusoidal endothelium

Figures

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  Fig. 1. Rg3-RGE significantly ameliorates inflammatory lesions of western diet mediated NAFLD. (A) The body weight during the whole experiment period. (B) The body weight on the last day. (C) The increased body weight ratio (%). (D) Gross observation of the liver. (E) The liver weight (g). (F) The liver to body weight ratio (%). (G) Representative images of H&E and Oil-Red O staining and Oil-Red O positive area (%). (H) Histopathological score of steatosis. (I) Lobular inflammation. (J) Ballooning degeneration and (K) NAFLD activity score. (L) The serum levels of ALT and (M) AST. Data are presented as the mean ± SD per experimental group. n = 5 mice in each group. ∗P < 0.05, ∗∗ P < 0.01, ∗∗∗P<0.001. Scale bars = 1 cm (D), 100 μm (G).

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  Fig. 2. Rg3-RGE inhibits the infiltration of inflammatory cells in liver parenchyma. (A) Representative immunohistochemistry images for myeloperoxidase (MPO) in liver parenchyma. (B) Number of MPO-positive cells. (C) Representative images of CD-68 immunofluorescences in liver. (D) CD-68-positive area (%). F-actin was used to visualize cell structures. (E) The results of qRT-PCR analysis for cxcl2, (F) cxcl1, (G) VCAM-1 and (H) CCL2. Data were normalized to mRNA expression levels of 18s. Data are presented as the mean ± SD per group n = 5 mice per group. ∗P < 0.05, ∗∗P<0.01. Scale bar, 100 μm (A and C).

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  Fig. 3. The Rg3-RGE significantly inhibits adhesion molecules' expression in LSECs. (A) Representative images of CD-206 and CD-68 immunofluorescence in liver. (B) The results of qRT-PCR analysis for TNF-α. (C) IL-1β, (D) CD-11b, (E) iNOS, (F) Arg 1, and (G) IL-10. Data were normalized to mRNA expression levels of 18s. (H) The protein expression of VCAM-1 in liver tissue. Data were normalized to protein expression level of β-actin. Data are presented as means ± SD per group. n = 4 to 5 mice per each group (A-H). ∗P < 0.05 and ∗∗P < 0.01. Scale bar, 100 μm (A).

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  Fig. 4. Rg3-RGE prevents the adhesion of inflammatory cells to CiGEnC. (A) Protein expression of ICAM-1 and VCAM-1 in CiGEnC. Data were normalized to the protein expression levels of β-actin. (B) Representative images of monocyte adhesion assay. The human monocytes (THP-1) were labelled by Calcein-AM and visualized in green. (C) Quantification of adherent human monocytes. Data are presented as the mean ± SD per group. Three independent experiments were conducted for the quantification (A-C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bar, 200 μm and 50 μm (B).

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  Fig. 5. Rg3-RGE significantly decreases the defenestration and VCAM-1 expression in LPS-treated primary liver sinusoidal endothelial cells (LSECs). (A) The procedure of mouse primary LSEC isolation. The figure was created with BioRender.com(B) The morphology of isolated LSECs. (C) Fluorescence-activated cell sorting (FACS) analysis for confirmation of isolated primary LSEC. (D) Effects of lipopolysaccharide (LPS) and Rg3-RGE treatment on the fenestrae structures and (E) number of fenestrae of primary LSECs. (F) The protein expression levels of VCAM-1 in LPS- and Rg3-RGE-treated primary LSECs. Data were normalized to the protein expression levels of β-actin. Data are presented as the mean ± SD per group. Three independent experiments were conducted for the quantification (C–F). ∗P < 0.05, and ∗∗P < 0.01. Scale bar, 50 μm (B). Scale bar, 2.5 μm (D).

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  Fig. 6. The scheme of the mechanism in the present study. Rg3-RGE attenuated NAFLD lesion by inhibiting inflammatory cell infiltrations by blocking VCAM-1. The figure was created with BioRender.com.