Ginsenoside Rb1-Enriched Saponin Fraction Inhibits M1 Macrophage Polarization by Suppression of TLR4 Trafficking in Metabolic Dysfunction-Associated Alcoholic Liver Disease

Background/Objectives: Metabolic dysfunction-associated alcoholic liver disease (MetALD) is a serious worldwide health concern, exhibiting metabolic dysfunction-associated lipid accumulation, alcohol-associated oxidative damage, and endotoxin-induced inflammation. Rb1-enriched red ginseng saponin fraction (RGSF) has been known to exhibit anti-inflammatory and anti-oxidative properties, but its role in MetALD remains to be fully elucidated. This study aims to investigate the specific mechanism of RGSF in the MetALD mouse model. Methods: The MetALD mouse model was administered with or without Rb1-RGSF for 7 weeks. Histopathological and molecular analyses, along with primary cell isolation, were conducted for in vivo and ex vivo investigations. M1 macrophage polarization was assessed by analyzing pro-inflammatory cytokine expression. NF-kB/p65 and TLR4 protein expression were measured before being visualized using immunofluorescence assays and confocal microscopy. Results: Histopathological examination revealed that RGSF treatment markedly reduced hepatic steatosis and attenuated inflammatory lesions in MetALD independent of oxidative stress. Notably, RGSF administration suppressed the LPS-induced internalization of surface TLR4. During the early inflammatory phase, RGSF prevented the LPS-mediated loss of the 130 kDa TLR4 form at the cell membrane, thereby limiting the generation of its 110 kDa cytoplasmic form. LPS-binding assay confirmed the direct interactions between TLR4 and RGSF. Conclusions: Collectively, these findings demonstrate that RGSF regulates TLR4 expression and trafficking, leading to the suppression of M1 macrophage polarization by inhibiting LPS–TLR4 surface interactions, thus exhibiting hepatoprotective effects.