Ginsenoside F2 Restrains Hepatic Steatosis and Inflammation by Altering the Binding Affinity of Liver X Receptor Coregulators

Figures

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  Fig. 1. GF2 reduces the LXR activity binding to the serine residue of the ligand binding pocket. (A) Heatmap showing expression of genes related to oxysterol synthesis pathway and LXR-mediated lipogenesis in liver tissues of NCD- and HFD-fed mice. (B) Relative LXRE luciferase activity in 293T cells treated in the presence or absence of GF2 and 10 μM T0901317. (C) Relative mRNA expression of Srebf1 and Abca1 in 293T cells treated with 10 μM T0901317 in the presence or absence of 1 μM GF2. (D) Protein tertiary structures of LXRα and LXRβ with amino acid residues in the binding pocket as predicted sites of GF2 binding. (E) Relative LXRE luciferase activity in 10 μM T0901317- and/or GF2-treated 293T cells following amino acid substitution at the predicted binding site of LXRα and LXRβ. HFD, high-fat diet; NCD, normal chow diet; LXRE, LXR element; GF2, Ginsenoside F2. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not statistically significant.

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  Fig. 2. GF2 maintains the binding affinity of LXRα corepressor (A, B) Schematic diagram of a measurement strategy for corepressor displacement (A) and coactivator recruitment (B) from LXR by applying TR-FRET assay. (C, D) TR-FRET ratio (520 nm/490 nm) of corepressor and coactivator binding to LXRα (C) or LXRβ (D) when treated with serially increasing doses of T0901017 or GF2 (upper panel) and with GF2 in the presence of 10 μM T0901317 (lower panel). Data are presented as mean ± SEM. The significance of all values was verified with respect to the value at the lowest concentration of drug treatment. *p < 0.05, **p < 0.01, ****p < 0.0001.

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  Fig. 3. LXRα-mediated lipogenesis is attenuated by GF2 in primary hepatocytes (A, B) Representative oil red O staining and quantification of primary hepatocytes after GF2 treatment with T0901317. Scale bar = 20 μm. (C) Relative mRNA expression level of Srebf1, Fasn, and Abca1, after GF2 treatment with 10 μM T0901317 on primary hepatocytes. (D, E) Relative mRNA expression level of Srebf1 and Fasn after GF2 treatment with 10 μM T0901317 on LXRα−/− (D) or LXRβ−/− (E) hepatocytes. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant.

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  Fig. 4. Inflammatory response of macrophages is attenuated by GF2 in LXRα dependent manner. (A) Relative mRNA expression level of LXRα (Nr1h3), LXRβ (Nr1h2), LXR target genes (Abca1, Cd36), and pro-inflammatory cytokines (Il1b, Tnf, and Il6) after PA treatment to M1-type BMDMs. (B, C) Relative mRNA expression level of Abca1 and proinflammatory cytokines after GF2 treatment with T0901317 in primed BMDM from WT (B) and LXRα−/− (C) mice. PA, palmitic acid; BMDM, bone marrow-derived macrophage. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not statistically significant.

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  Fig. 5. GF2 alleviates MASLD by downregulating lipogenesis and inflammatory response. Physiological and molecular analyses of livers from WT mice fed with HFD for 12 weeks with or without GF2 administration (n = 6/group). (A) Representative H&E and oil red O (ORO) staining in liver sections. Scale bar = 100 μm. (B) Hepatic triglyceride level, (C) blood glucose level in glucose tolerance test (GTT) and insulin tolerance test (ITT). (D) Relative mRNA expression level of Nr1h3, N1rh2, and lipogenesis-related LXR target genes (Srebf1 and Fasn). (E) Immunoblot of SREBP1 (FL, Full length; M, mature form) and FASN. (F) Immunofluorescence staining of hepatic macrophages with F4/80 in frozen liver tissue. Scale bar = 100 μm. (G) Relative mRNA expression level of Il1b, Tnf, and Il6 in liver mononuclear cells (MNCs). TG, triglyceride; LPF, low power fields. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 6. LXRα deficiency abolishes the effect of GF2 in MASLD. Physiological and molecular analyses of livers from LXRα−/− mice fed with HFD for 12 weeks with or without GF2 administration (n = 4/group). (A) Body and liver weight of mice. (B) Representative liver gross, H&E, and oil red O staining in liver sections. Scale bar = 100 μm. (C) Hepatic and serum triglyceride levels (D) Relative mRNA expression level of Srebf1 and Fasn. (E) Cell number per g liver of hepatic macrophages, neutrophils, and eosinophils. (F) Relative mRNA expression level of Il1b, Tnf, and Il6 in liver mononuclear cells (MNCs). Data are presented as mean ± SEM. ns, not statistically significant.