Fermented Korean Red Ginseng Extract Enriched in Rd and Rg3 Protects against Non-Alcoholic Fatty Liver Disease through Regulation of mTORC1

Figures

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  Scheme 1. Extract preparation of solid-state fermented of Red Ginseng (RG) by Cordyceps militaris.

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  Figure 1. Fermented RG with Cordyceps militaris (CRG) treatment prevents inflammation and injury in fast-food diet (FFD)-induced non-alcoholic fatty liver disease (NAFLD). CRG was orally administered to mice for the last 8 weeks till the animals were sacrificed. (A) Each section was stained with H&E. The protein levels of (B) Il-1β; (C) Ccl2 and (D) Ccl5 in liver tissue lysates were determined by ELISA. Expressions of (E) Ccl2 and (F) Ccl5 in liver tissue were measured by qRT-PCR shown as fold change compared with normal chow diet (NCD) mice. The level of (G) alanine aminotransferase (ALT) and (H) aspartate aminotransferase (AST) in the serum were measured. Original magnification, X 20. * p < 0.05 versus vehicle (Veh), ** p < 0.01 versus Veh, *** p < 0.001 versus Veh.

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  Figure 2. CRG treatment prevents hepatic steatosis in FFD-induced NAFLD. (A) Each section was stained with BODIPY to determine lipid accumulation. (B) Hepatic triglyceride (TG) level was measured. Expression of fatty acid synthesis genes such as (C) Acc and (D) Fas; and fatty acid oxidation genes (E) Cpt1 and (F) Acox were evaluated by qRT-PCR shown as fold change compared with NCD diet mice. Original magnification, X 20. ** p < 0.01 versus vehicle (Veh), *** p < 0.001 versus Veh, **** p < 0.0001 versus Veh.

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  Figure 3. Fermentation of RG with C. militaris enhances the protective effect on lipid accumulation and inflammation. (A) Lipid accumulation was assessed by BODIPY staining. Expression of fatty acid synthesis genes (B) Fas; (C) Srebp-1c and fatty acid oxidation genes marker (D) CD36; and (E) Acsl determined by qRT-PCR shown as fold change compared with Bovine serum albumin (BSA). The expression of inflammatory genes (F) Ccl2; (G) Ccl5; (H) Tnf-α and (I) iNos was measured by qRT-PCR. Original magnification, X 20. * p < 0.05 versus vehicle (Veh), ** p < 0.01 versus Veh, *** p < 0.001 versus Veh.

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  Figure 4. Ultra-high-performance liquid chromatography (UPLC) chromatogram of ginsenosides in (A) RG and (B) CRG. The first red box is Rd, the second one is Rg3.

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  Figure 5. The chemical structure of (A) Rd; (B) Rg3s, and Rg3r.

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  Figure 6. Rd and Rg3 protect mitochondria-mediated lipotoxic apoptosis. (A) Hep3B cells were measured for MitoSOX 12 h after treatment with H2O2 and CRG. (B) Hep3B cells were transfected with pLVX-puro-mt-Keima. Analysis of expressing mt-Keima treated with ginsenoside Rd for 24 h. Rd mediated mitophagy was quantified by flow cytometry with 2 μM CCCP. The excitation wavelength is 586 nm in neutral and 440 nm in acidic environment. (C) PA-induced cytotoxicity was measured by LDH. (D) Representative western blotting analysis of Bcl-2. CRG 62.5 μg/mL is referred to as CRG-H and CRG 31.25 μg/mL as CRG-L. * p < 0.05 versus vehicle (Veh), ** p < 0.01 versus Veh, *** p < 0.001 versus Veh.

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  Figure 7. Rd and Rg3 -induced anti-steatotic effect is dependent on mTORC1. (A–C) Primary hepatocytes were incubated with PA (125 μM) and WY14643 (30 μM) for 6 h. The expression of PPARα target genes (A) Acox; (B) Acsl and (C) Hmgcs2 were determined by qRT-PCR. (D) The liver tissue and (E) Hep3b cells were incubated with PA (200 μM) and CRG for 24 h. (F) Primary hepatocytes were incubated with PA (200 μM) and Rapamycin (50 nM) with 2.5 μg/mL Rd or 5 μM Rg3 for 24 h. Lipid accumulation was assessed by BODIPY staining. Original magnification, X 20. * p < 0.05 versus vehicle (Veh), ** p < 0.01 versus Veh, *** p < 0.001 versus Veh, **** p < 0.0001 versus Veh.

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  Figure 8. Rd and Rg3 exert anti-inflammatory activity via mTORC1-mediated M2 polarization. (A) Raw264.7 cells were incubated with LPS and CRG for 24 h. (B–I) RAW264.7 cells were pretreated with Rd or Rg3 for 3 h and then stimulated with LPS for 6 h. Expression of M1 marker genes (B) Ccl2; (C) Ccl5; (D) Il-1β; (E) Il-6; (F) iNos and (G) Tnf-α and (C) M2 marker genes (H) CD163 and (I) Il-10 were measured by qRT-PCR. * p < 0.05 versus vehicle (Veh), ** p < 0.01 versus Veh, *** p < 0.001 versus Veh, **** p < 0.0001 versus Veh.