Seoung-Woo Lee,Su-Min Baek,Young-Jin Lee,Tae-Un Kim,Jae-Hyuk Yim,Jun-Hyeok Son,Hee-Yeon Kim,Kyung-Ku Kang,Jong Hun Kim,Man Hee Rhee,Sang-Joon Park,Seong-Kyoon Choi,Jin-Kyu Park
Ginsenoside Rg3-enriched Korean Red Ginseng extract attenuates Non-Alcoholic Fatty Liver Disease by way of suppressed VCAM-1 expression in liver sinusoidal endothelium
chemotaxis,ginsenoside,liver sinusoidal endothelium,NAFLD,VCAM-1
Background The incidence and clinical importance of nonalcoholic fatty liver disease (NAFLD) has emerged. However, effective therapeutic strategies for NAFLD have yet to be found. Panax ginseng (P. ginseng) is a traditional herb in Eastern Asia with therapeutic effects in many chronic disorders. However, the precise effects of ginseng extract on NAFLD are currently unknown. In present study, the therapeutic effects of Rg3-enriched red ginseng extract (Rg3-RGE) on the progression of NAFLD were explored. Methods Twelve-week-old C57BL/6 male mice were fed a chow or western diet supplemented with high sugar water solution with or without Rg3-RGE. Histopathology, immunohistochemistry, immunofluorescence, serum biochemistry, western blot analysis, and quantitative RT-PCR were used for in vivo experiment. Conditionally immortalized human glomerular endothelial cell (CiGEnC) and primary liver sinusoidal endothelial cells (LSECs) were used for in vitro experiments. Results Eight weeks of Rg3-RGE treatment significantly attenuated the inflammatory lesions of NAFLD. Furthermore, Rg3-RGE inhibited the inflammatory infiltrate in liver parenchyma and the expression of adhesive molecules to LSECs. Moreover, the Rg3-RGE exhibited similar patterns on the in vitro assays. Conclusion The results demonstrate that Rg3-RGE treatment ameliorates NAFLD progression by inhibiting chemotaxis activities in LSECs.
Ginsenoside Rg3-enriched Korean Red Ginseng extract attenuates Non-Alcoholic Fatty Liver Disease by way of suppressed VCAM-1 expression in liver sinusoidal endothelium

The incidence and clinical importance of nonalcoholic fatty liver disease (NAFLD) has emerged. However, effective therapeutic strategies for NAFLD have yet to be found. Panax ginseng (P. ginseng) is a traditional herb in Eastern Asia with therapeutic effects in many chronic disorders. However, the precise effects of ginseng extract on NAFLD are currently unknown. In present study, the therapeutic effects of Rg3-enriched red ginseng extract (Rg3-RGE) on the progression of NAFLD were explored.

Twelve-week-old C57BL/6 male mice were fed a chow or western diet supplemented with high sugar water solution with or without Rg3-RGE. Histopathology, immunohistochemistry, immunofluorescence, serum biochemistry, western blot analysis, and quantitative RT-PCR were used for in vivo experiment. Conditionally immortalized human glomerular endothelial cell (CiGEnC) and primary liver sinusoidal endothelial cells (LSECs) were used for in vitro experiments.

Eight weeks of Rg3-RGE treatment significantly attenuated the inflammatory lesions of NAFLD. Furthermore, Rg3-RGE inhibited the inflammatory infiltrate in liver parenchyma and the expression of adhesive molecules to LSECs. Moreover, the Rg3-RGE exhibited similar patterns on the in vitro assays.

The results demonstrate that Rg3-RGE treatment ameliorates NAFLD progression by inhibiting chemotaxis activities in LSECs.

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of chronic metabolic syndrome [1]. NAFLD is generally caused by excessive lipid accumulation in the liver parenchyma (>5−10% of total liver volume) without alcohol intake, viral infection, or exposure to toxins [[2], [3], [4]]. The pathogenesis includes diet types (i.e., high-fat or high-carbohydrate diet), obesity, microbiota, imbalance of lipid homeostasis, excessive lipid accumulation and inflammatory cell infiltration in the parenchyma [5]. NAFLD ranges from steatosis to nonalcoholic steatohepatitis (NASH) which can eventually induce irreversible hepatic injures, such as cirrhosis and hepatocellular carcinoma (HCC) [6]. The increasing incidence of sedentary and westernized lifestyles on a global scale recently have contributed to rising trends in the NAFLD progression [7]. Consequently, the global incidence of NAFLD has steadily emerged, with NAFLD becoming the most common chronic liver disease [8]. Thus, research focused on developing treatments for NAFLD lesions has intensified [[9], [10], [11]]. Unfortunately, the specific pathological mechanism and therapeutics for NAFLD remain unclear. Panax ginseng (P. ginseng) is a well-known traditional herb in Eastern Asia, where the roots of P. ginseng are commercially produced for traditional medicine and used as a remedial herb in Korea, Japan, and China [12,13]. Due to its anti-inflammatory, antioxidant, and health-enhancing properties, interest on the P. ginseng in various diseases has increased [14]. The pharmacological activities of P. ginseng mainly originate from ginsenoside, which belongs to natural steroid glycosides, and triterpene saponins [15]. Among these ginsenosides, ginsenoside Rg3 has received attention due of its high physiological effects [16]. Additionally, previous studies have demonstrated therapeutic roles of ginsenoside Rg3 in several chronic metabolic disorders [17]. Despite extensive studies, the precise therapeutic effects and protective mechanisms of P. ginseng derivative, including ginseng extracts and ginsenoside Rg3, on NAFLD remain elusive. In this study, comprehensive experiments were performed to elucidate the role of ginseng derivative on NAFLD using ginsenoside Rg3-enriched red ginseng extracts (Rg3-RGE)

Rg3-RGE was acquired following previously described methods [18]. Briefly, Rg3-RGE was provided by Korea Ginseng Corporation (Daejeon, Korea). The stem and root of Korean Red Ginseng (25% of stem and 75% of root) were extracted with water and 55% ethanol. The extract was concentrated by multiple extraction and analyzed by high performance liquid chromatography (HPLC). The resulting extract's constituent was composed of 20(S)-Rg3 (44.91mg/g), Rb1 (3.86mg/g), Rc (1.20mg/g), Rb2 (1.53mg/g), Rd (1.6mg/g), Rf (1.28mg/g), Rh1 (3.71mg/g), 20(S)-Rg2 (3.55mg/g), and 20(R)-Rg3 (6.78mg/g) with total 67.41mg/g.

Twelve-week-old male C57BL/6 mice (n = 15) were separated into three groups (n = 5 per group): (1) control group (Chow) fed a chow diet (SAFE D40; Safe Diet; Rosenberg, Germany); (2) WD group fed a western diet (D12079B; Research Diets, New Brunswick, NJ, USA) and sugar water solution (23.1 g/L fructose + 18.9 g/L glucose in 1L filtered tap water) (3) WD + Rg3-RGE group fed a western diet with Rg3-RGE (5 mg/kg)-supplemented sugar water solution in the last 8 weeks of the experiment. The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) at Kyungpook National University (approval number 2021-0105).

The liver tissue samples were fixed in 10% neutral buffered formalin and were embedded in paraffin wax for pathologic examination. The paraffine block were section into 4-μm-to 5-μm thickness for histopathologic analysis including hematoxylin and eosin (H&E) staining and immunohistochemistry. The 4% paraformaldehyde fixed tissue samples were rinsed three times with PBS. After then, the tissues were immersed in 30% sucrose diluted in PBS and embedded with frozen OCT medium. The samples were sectioned into 3-μm-to 10-μm-thickness for immunofluorescence and Oil-Red-O staining. A histopathological NAFLD grading was obtained following NASH Clinical Research Network (CRN) grading system [19]. All histopathological scores were graded from at least five random × 200 fields for each sample. Oil-Red O positive areas were quantified with imageJ (NIH, Bethesda, MD, USA) program.

Immunohistochemistry was performed as previously described [20]. Antigen retrieval was performed in 3% hydrogen peroxide diluted in methanol, followed by steaming in citric acid buffer. After cooling down for 2 h at RT, the slides were cleansed in distilled water and PBS before incubating in blocking solution (Life Technologies, Frederick, MD, USA). After blocking, the slides were adapted to primary antibody including anti-MPO (PA5-16672; Thermo Fisher Scientific, Waltham, MA, USA). After overnight incubation, the sections were rinsed three times in PBS and incubated with a secondary antibody and streptavidin-HRP (Life Technologies, Frederick, MD, USA). Then, the slides were visualized by diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA, USA) and 10% hematoxylin was used for counterstaining.

The slides were washed in PBS and 0.1% TritonX-100 diluted in 1X PBS was used for permeabilization. Subsequently, the slides were rinsed three times in PBS, blocked with 5% donkey serum and were incubated primary antibody diluted in 5% donkey serum. Anti-CD68 (MCA1957GA; Bio-Rad), anti-CD206 (AF2535; R&D Systems, Minneapolis, MN, USA) were used. Alexa Fluor 555 donkey anti-goat IgG (ab150134; Abcam, Cambridge, MA, USA) and alexa fluor 488 donkey anti-rat (ab150153, Abcam) were used as secondary antibody. ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology) was used for mounting.

The blood samples were incubated at RT for clotting. The resulting clots were removed by centrifuging at 3000 rpm for 15 min at 4°C. Alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were analyzed by an automated analyzer (TBA-120FR; Toshiba, Tokyo, Japan).

Western blot procedures were performed as described previously [20]. The protein concentration for homogenized liver lysates was evaluated with DC Protein Assay Kit (Bio Rad, Hercules, CA, USA). The protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After then, the protein was transferred to a PVDF membrane (IPVH00010; Millipore, Billerica, MA, USA). 5% skim milk diluted in tris-buffered saline with 0.1% Tween-20 (TBS-T) was used as a blocking reagent. For primary antibodies, anti-ICAM-1 (sc-7891; Santa Cruz Biotechnology), anti-VCAM-1 (ab134047; Abcam), and anti-β-actin (A1978; Sigma, St. Louis, MO, USA; and sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. After then, the membrane was rinsed three times in TBS-T and incubated with secondary antibody including goat-anti-rabbit (401393; Calbiochem, San Diego, CA, USA) or goat-anti-mouse (401253; Calbiochem) antibody. The membranes were developed with ECL solution (Translab, Seoul, Korea) and were exposed to Amersham™ Imager 680 (GE Healthcare, Uppsala, Sweden) or ImageQuant LAS 500 imager (GE Healthcare). For the internal standard, β-actin (Sigma) was used.

Total liver RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The cDNA synthesis was performed with the RT Prime kit (EBT-1520; ELPIS Biotech, Daejeon, Korea). For qRT-PCR, SYBR Green with low ROX (RT500M; Enzynomics, Daejeon, Korea) and primers (5 pmol of each forward and reverse primer) were mixed with cDNA samples. 18S ribosomal RNA was used as an internal standard to normalize mRNA expression. The followings are primers used in the study [[21], [22], [23], [24], [25], [26], [27], [28], [29]]: 18s (Forward: ACGGAAGGGCACCACCAGGA; Reverse: CACCACCACCCACGGAATCG), TNF-α (Forward: AGGCTGCCCCGACTACGT; Reverse: GACTTTCTCCTGGTATGAGATAGCAAA), IL-1β (Forward: CTTTGAAGTTGACGGACCC; Reverse: TGAGTGATACTGCCTGCCTG), IL-10 (Forward: GCCTTGCAGAAAAGAGAGCT; Reverse: AAAGAAAGTCTTCACCTGGC), CD-11b (Forward: GTGTCCGCAAGAACACCAAG; Reverse: GGACAGGGTCTAAAGCCAGG), cxcl1(Forward: CTGGGATTCACCTCAAGAACATC; Reverse: CAGGGTCAAGGCAAGCCTC) , cxcl2 (Forward: GAAGTCATAGCCACTCTCAAGG; Reverse: CCTCCTTTCCAGGTCAGTTAGC), CCL2(Forward: GTGGGGCGTTAACTGCAT; Reverse: CAGGTCCCTGTCATGCTTCT), VCAM-1 (Forward: TCATCCCCACCATTGAAGAT; Reverse: ACCTAGCGAGGCAAACAAGA), ICAM-1 (Forward: ATTCGTTTCCGGAGAGTGTG; Reverse: CAGCACCGTGAATGTGATCT), iNOS (Forward: AATCTTGGAGCGAGTTGTGG; Reverse: CAGGAAGTAGGTGAGGGCTTG), and Arg1 (Forward: CTCCAAGCCAAAGTCCTTAGAG; Reverse: GGAGCTGTCATTAGGGACATC).

CiGEnCs were seeded in a six-well plate (300006; SPL Life Sciences, Korea) at a density of 1 × 105 cells per well. Human endothelial cell growth medium was used for culture. From day 1 after seeding, the cells were treated with 25 and 100 μg/ml of sterile filtered Rg3-RGE diluted in PBS and 1 μg/ml of LPS (L2880; Sigma-Aldrich, St Louis, MO, USA) or LPS only as a control for 24 h. These samples were harvested in 1x sample buffer.

CiGEnCs were incubated in human endothelial cell growth medium at 90‒95% confluence on 12-well plates (300012; SPL Life Sciences). The cells were treated with Rg3-RGE and LPS or LPS only for 24 h. THP-1 cells were labeled with 50 μM Calcein-AM (C3100MP; Thermo Fisher Scientific) in RPMI 1640 medium supplemented with 10% FBS and 25 mM HEPES for 30 min. The calcein-labelled THP-1 cells were then washed twice with DPBS (SH30256.01; Cytiva) and incubated at 37°C, 5% CO2 for 1 h. Then, the calcein-labelled THP-1 cells were seeded at a density of 1 × 105 cells/well onto CiGEnCs and incubated for 1 h with gentle shaking every 15 min. After then, non-adherent THP-1 cells were removed by three-times wash in PBS.

Primary LSECs were isolated from C57BL/6 mice using a modified method of a previously reported protocol [30]. A cell suspension, which was obtained after collagenase perfusion, was centrifuged at 50 × g for 5 min at 4°C to isolate the non-parenchymal cell fraction (NPC) from the hepatocyte fraction. The supernatants were centrifuged at 350 × g for 10 min at 4°C to obtain the NPC pellets. The resulting pellets were re-suspended in endothelial cell growth basal medium (EBM)-2 (CC-3162; Lonza Clonetics, Walkerville, MD, USA). These NPC cell suspensions were centrifuged at 900 × g at 4°C for 20 min after the density gradient by using 25% and 50% Percoll™ solution (17-0891-02; GE Healthcare). The LSECs were seeded on to collagen-coated six-well plates in EBM-2 supplemented with 15% FBS (16000044; Gibco), 1% streptomycin/penicillin/amphotericin B (LS-203-01; Welgene, Gyeongsan, Republic of Korea), and 1% L-glutamine (Welgene). At 4, 24, and 48 h after cell culture, the LSECs were analyzed by scanning electron microscopy, fluorescence-activated cell sorting (FACS) analysis, and western blot analysis. The animal experiment and procedure for primary cell isolation were approved by IACUC at Kyungpook National University (approval number 2022-0299).

After 24 h of seeding, the primary LSECs were collected with 0.025% Trypsin-EDTA diluted in 1 × DPBS and centrifuged at 3000 rpm for 3 min. The resulting cell pellets were washed with FACS buffer (2% FBS diluted in 1 × DPBS). Next, the cells were centrifuged again at 3000 rpm for 3 min. The cell pellets were fixed with 4% PFA for 20 min at 4°C. After fixation, 500 μl of FACS buffer was added, and the solution was centrifuged at 3000 rpm for 3 min. The cell pellets were washed with FACS buffer three times. The cell pellets were then divided into the two new 1.5-ml microtubes for the analysis of an unstained negative sample and an anti-CD68 (Bio-Rad) and anti-eNOS (NB300-500; Novus Biologicals, Centennial, CO, USA) double-stained sample. 0.1% Triton-X was used for permeabilization for the both samples. The samples were washed with 500 μl of FACS buffer, and centrifuged at 3500 rpm for 5 min. For the staining group, the cells were stained with anti-CD68 (1:100) and eNOS (1:50) for 30 min at 4°C. For the unstained sample, only FACS buffer was added. Then, the cells were washed with 500 μl of FACS buffer at 3000 rpm for 5 min. The double-stained cells were resuspended with 500 μl of FACS buffer with the secondary antibody (Alexa Fluor 488 donkey anti-rabbit IgG (A-21206; Thermo Fisher Scientific) (1:500) for eNOS and Alexa Fluor 647 donkey anti-rat IgG for CD68 (ab150155; Abcam) (1:500) at 4°C in the dark for 30 min. Lastly, the cells were rinsed with 500 μl of FACS buffer and centrifuged at 3500 rpm for 5 min. The cells were resuspended in 500 μl of ice cold FACS buffer. The resulting cells were subjected to FACS analysis.

After 4 h of seeding, the primary LSECs were treated with media only, media with LPS (1 μg/ml), or media with LPS and Rg3-RGE (100 μg/ml) for 8 h. After treatment, the cells were washed with 1 × DPBS and fixed with a solution composed of 2% PFA/2.5% Glutaraldehyde (27372S-1230; Junsei, Tokyo, Japan) for 2 h at RT. The fixed cells were washed with 1 × PBS twice for 5 min. Then, the cells were secondarily fixed with 1% Osmium (VIII) oxide (5741-4101; Duksan, Gyeonggi, Republic of Korea) diluted in distilled water for 2 h at RT. The cells were then washed with 1 × PBS twice for 5 min. The washed cells were dehydrated with 30%, 50%, 70%, 80%, 95%, and 100% of ethanol for 10 min per step. The ethanol was dried at RT and the samples were coated with platinum.

All data are presented as mean ± standard deviation (SD) with statistical significance considered at NS (not significant, P > 0.05), ∗ (P < 0.05), ∗∗ (P < 0.01) and ∗∗∗ (P < 0.001). The normal distribution of data was verified by D'Agostino-Pearson normality test. Statistical analysis for the data was evaluated by Kruskal-Wallis one-way analysis of variance (ANOVA), Mann-Whitney U test and unpaired Student's t-test. All statistical analysis was conducted using GraphPad InStat 3.0 (GraphPad Software Inc., San Diego, CA, USA) and GraphPad Prism 5.0 (GraphPad Software Inc).

For entire experimental periods, WD + Rg3-RGE group exhibited relatively decreased body weight gain compared than WD group (Fig. 1A). Therefore, body weight and an increased body weight ratio were generally decreased in WD + Rg3-RGE group compared to WD group. (p = 0.07) (Fig. 1B and C). Overall, the livers of the WD-fed mice groups exhibited an increased size, a pale surface, and multifocal white spots, suggesting hepatic lipid accumulation, compared to the mice fed a chow diet (Fig. 1D). However, WD + Rg3-RGE group exhibited decreased pattern of liver weight and liver weight to body weight ratio compared to WD mice group. (Fig. 1E and F). Following microscopic examinations indicated that the WD group exhibited notably increased steatosis lesions compared to the control group, indicative of NAFLD progression (Fig. 1G). Interestingly, the WD + Rg3-RGE group exhibited a decreasing tendency of steatosis lesions compared to WD group consistent with body and liver weight results (Fig. 1G and H). Moreover, WD + Rg3-RGE group exhibited significantly decreased scores for lobular inflammation and ballooning degenerations and NAS scores (p = 0.06) compared to WD group (Fig. 1I–K). Following serum biochemical assay revealed that the ALT and AST serum levels were significantly decreased by RG3-RGE treatment. (Fig. 1L and M). These data suggest that the Rg3-RGE significantly attenuated the inflammatory lesions of NAFLD in WD-fed mice.

Considering that Rg3-RGE treatment significantly inhibited NAFLD-mediated inflammatory lesions, the infiltrations of inflammatory cells in liver parenchyma were examined in all mice groups. The results of immunohistochemistry assays revealed that 16 weeks of western diet treatment markedly increased the MPO (a neutrophil marker)-positive cells, while the WD + Rg3-RGE group demonstrated a significantly decreased MPO-positive cells compared to the WD group. (Fig. 2A and B). Subsequently, immunofluorescence staining experiments revealed that the CD-68 (a marker of macrophages) antibody-positive area (%) was also significantly decreased by Rg3-RGE treatment (Fig. 2C and D). Consistent with these histopathological results, qRT-PCR results showed that the WD + Rg3-RGE group exhibited notably decreased patterns of mRNA levels involved in the chemoattractant and adhesion pathways in inflammatory cells (Fig. 2E–H). These results indicate that Rg3-RGE treatment significantly inhibited the infiltration of inflammatory cells into hepatic parenchyma.

Next, we investigated the polarization of intrahepatic macrophages, which is a crucial factor for the regulation of hepatic inflammatory responses. We analyzed the ratio of M1/M2 macrophages in the liver by visualizing M2 macrophages via colocalization with CD-68 and CD206 (markers of whole macrophages and M2 macrophages) (Fig. 3A). The immunofluorescence results indicated that the ratio of M1/M2 polarization was similar between the WD and WD + Rg3-RGE groups. Subsequently, qRT-PCR experiments showed that the levels of mRNA associated with pro- and anti-inflammatory responses were not significantly affected by Rg3-RGE treatment, except iNOS (Fig. 3B–G). However, the immunoblotting analysis showed significantly decreased protein expression of VCAM-1, which is an essential molecule for inflammatory cell adhesion to LSEC, in the WD + Rg3-RGE group compared to the WD group (Fig. 3H). These data indicate that the Rg3-RGE may attenuate liver injuries via suppressing expression of adhesion molecules in LSEC rather than regulating polarization of macrophages.

To confirm the effects of Rg3-RGE on vascular endothelium, in vitro analyses using CiGEnCs were performed. In vitro experiments revealed that the inflammatory cell adhesion molecules (e.g., VCAM-1 and ICAM-1) were significantly upregulated by LPS treatment. However, Rg3-RGE treatment significantly decreased the VCAM-1 and ICAM-1 expression compared to the LPS-treated control group (Fig. 4A). Next, we investigated whether Rg3-RGE inhibited the adhesion of inflammatory cells using in vitro analysis. Subsequent experiments demonstrated that the adhesion of THP-1 cells to CiGEnCs was dose-dependently decreased by Rg3-RGE treatment. (Fig. 4B and C). These results demonstrate that Rg3-RGE treatment inhibited the adhesion of inflammatory cells to vascular endothelium.

To determine whether the phenotype was reproduced in LSECs, primary LSECs were isolated from C57BL/6 male mice (Fig. 5A and B). To confirm whether the isolated cells were LSECs, FACS analysis was performed; 72.1% of total stained cells were found to be pure eNOS-positive LSECs (Fig. 5C). Using these LSECs, the effect of RG3-RGE on LSEC fenestrae was investigated. While LPS-treated LSECs showed a significantly decreased number of fenestrae compared to those of control group, fenestrae in LPS with Rg3-RGE-treated cells showed a significantly increased number of fenestrae, indicative of less capillarization (Fig. 5D and E). Interestingly, VCAM-1, which is expressed in endothelial cells after pro-inflammatory stimulation, was also found to be increased nearly by a 1.5-fold in LPS-treated LSECs but significantly decreased in the Rg3-RGE-treated group, highlighting the anti-inflammatory role of Rg3-RGE (Fig. 5F).

In present study, we investigated the therapeutic effects and mechanisms of Rg3-RGE treatment against Western diet-mediated NAFLD lesions. WD + Rg3-RGE treated group showed a decreasing pattern in liver weight, liver/body weight ratio, and steatosis scores (Fig. 1). Moreover, WD + Rg3-RGE treated group exhibited generally well retained liver architectures and significantly lower inflammatory grades when compared to WD group (Fig. 1). Considering that inflammation could be the cause of lipid accumulation due to the stress response of hepatocytes, and the distortion of hepatic structures is a signal of hepatic injuries, Rg3-RGE may protect hepatocytes from western diet-mediated NAFLD lesions [31,32]. Serum biochemical assays also showed a significant alleviation of ALT and AST in the WD + Rg3-RGE group compared to WD group (Fig. 1). Consequently, immunohistochemistry and immunofluorescence staining revealed that the average number of infiltrated inflammatory cells in liver parenchyma were significantly decreased by Rg3-RGE treatment (Fig. 2). Thus, we attempted to investigate the polarization of macrophages, which are key immune regulators of NAFLD [33,34]. However, unexpectedly, the ratio of M1/M2 macrophages and the gene expression associated with M1 and M2 macrophage were not affected by Rg3-RGE treatment (Fig. 3). This raises the question of how Rg3-RGE protects the liver from NAFLD-mediated inflammatory responses without affecting macrophage polarization in the liver. Based on the reduced number of inflammatory cells, we focused on the LSECs. LSECs are highly specialized endothelial cells in the liver involved in cellular crosstalk between hepatocytes and hepatic non-parenchymal cells [35]. LSECs are the most abundant hepatic non-parenchymal cell type in the liver, accounting for 15-20% of total liver cells [36]. LSECs play a pivotal role in the recruitment and adhesion of immune cells to liver parenchyma by expressing various chemokines and immune cell adhesive molecules [[37], [38], [39]]. Remarkably, WD + Rg3-RGE group exhibited significantly decreased gene and protein expression levels involved in chemotactic response of inflammatory cells (Fig. 2, Fig. 3). Subsequent in vitro experiments demonstrated that Rg3-RGE treatment inhibited LPS-mediated activation of CiGEnC cells and inhibited the adhesion of inflammatory cells to endothelial cells (Fig. 4). These patterns were also reproduced in isolated primary mouse LSECs (Fig. 5). Interestingly, Rg3-RGE-treated LSECs showed significantly increased number of fenestrae with well-retained structure (Fig. 5). Fenestrae are pores ranging 50-200 nm in diameter, and their organization can be changed by different stimuli to effectively scavenge macromolecules, such as lipids and lipoproteins [40]. In the early onset of NAFLD, LSECs play anti-inflammatory roles via the downregulation of pro-inflammatory chemokine secretion [40]. However, as NAFLD progresses to NASH, LSECs lose fenestrae structure and form a basement membrane through a process referred to as “capillarization”, a well-known feature of NASH [40,41]. After capillarization, LSECs are considered to be in a dysfunctional state, in which LSECs exhibit pro-vasoconstrictive, pro-inflammatory, and pro-fibrotic features, resulting in the absence of liver regeneration [42]. Moreover, capillarized LSECs enhance cellular adhesion molecules expressions including VCAM-1 or ICAM-1, to recruit blood leucocytes, leading these leucocytes to enter the liver parenchyma, corresponding to the most severe phenotype, which was only observed in the WD group. Taken together, we can conclude that Rg3-RGE treatment attenuated NASH lesions by selectively inhibiting inflammation via ameliorating the adhesion of inflammatory cells to LSECs. In the current study, Rg3-RGE significantly inhibited the adhesion and recruitment of inflammatory cells to mouse LSECs and CiGEnCs. CiGEnC exhibit similar gene expression patterns as human umbilical cord endothelial cells (HUVECs) [43]. Considering that HUVECs are widely used as a model of human vascular endothelium, Rg3-RGE may not only have potential positive effects for LSECs but also act as a therapeutic agent in many other organs [[44], [45], [46]]. Consistently, Rg3 in inhibition of adhesion of inflammatory cells to HUVEC and expression of adhesion molecules in mouse aorta was reported [47]. Additionally, it was reported that Rg3 treatment notably attenuated ox-LDL-mediated atherosclerosis, and while Rg3 significantly improve heart failure lesions [48,49]. Moreover, other components of the ginsenoside used in this study, for example, Rb1 or Rg2 were also reported to inhibit inflammatory and adhesion molecules in atherosclerosis [50]. However, the role of ginsenoside in inhibiting the recruitment and attachment of inflammatory cells to the LSECs and the related mechanism have not been suggested yet. We demonstrated that the therapeutic effects of Rg3-RGE in western-diet mediated NAFLD via inhibiting adhesion of inflammatory cells to vascular endothelium. However, there are still limitations that need to be resolved. First, in the present study, we suggested that the Rg3-RGE significantly attenuated NASH lesions via inhibiting inflammatory cell adhesion to LSECs. Unfortunately, the precise mechanisms that relate in Rg3-RGE application and decreased expressions of vascular adhesion molecules are still unknown. Considering VCAM-1 is significantly increased with LPS treatment in endothelial cells, it is assumed that Rg3-RGE might have an inhibitory role in LPS associated mechanism such as TLR4 pathway. Second, we provided Rg3-RGE with the dose of 5mg/kg daily to mice. The dose was enough to alleviate inflammatory lesions and serum ALT and AST levels, but the dose seems to be not sufficient to alleviate steatosis lesion significantly between WD and WD + Rg3-RGE groups although a decreasing tendency was observed. For the reason, a higher dose of Rg3-RGE needs to be adapted in the further study. To summarize, Rg3-RGE was found to significantly ameliorate western diet-mediated NAFLD lesions in mice by inhibiting the adhesion of inflammatory cells to the vascular endothelium. (Fig. 6). Subsequent in vitro experiments revealed that Rg3-RGE inhibited the adhesion of inflammatory cells to LSECs. These results provide novel insights into the role of ginsenoside in NAFLD via suppression of VCAM-1 expression in LSECs.

SWL and SMB designed the experiment, performed experiment and data interpretation, and wrote the manuscript. YJL, TUK, JHY, JHS, HYK and KKK were involved in data collection. JHK, MHR and SJP were involved in review of the manuscript. SKC and JKP designed the concept of the project, supervised the entire research progress within data collection, analysis, and interpretation, reviewed and revised the manuscript. Each author read and approved the final version of the manuscript.

This work was supported by the 2020 grant from 10.13039/501100015826The Korean Society of Ginseng, the 10.13039/501100010274DGIST R&D Program of the 10.13039/501100014188Ministry of Science and ICT (2020010096), and National Research Foundation of Korea (NRF) of the Ministry of Education (2019R1I1A2A01060031).

The authors declared that they had no conflicts of interests.