• The Journal of Internal Korean Medicine
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• v.31 no.2
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• pp.314-330
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• 2010

Objective : This study was performed to investigate the anti-fibrogenic effect and changes of inflammation-related genes by YBR I and YBR II (YBR I: Arteisiae Capillaris Herba, Atractylodis Rhizoma Alba, Hoelen/ YBR II: YBR I +Sanguisorbae Radix, Biotae Cacumen, Cirsii Japonici Herba) on HSC(hepatic stellate cells)-T6 and TAA-induced rat liver tissue. Materials and Methods : HSC-T6 were treated with various concentrations of distilled-water extract YBR I and YBR II extract for 24, 48 and 72 hours. After the treatment, cell viability, proliferation, procollagen levels and IL-6 levels were measured by using MTT Assay, BrdU Assay, Procollagen Type 1 C-peptide EIA kit, and Murine IL-6 ELISA Development kit. Rat liver fibrosis was induced by intraperitoneal TAA injection of 150mg/kg 3 times a week for 6 weeks. After the treatment, body weight, liver & spleen weights, liver function test, complete blood cell count and change of portal pressure were studied. In addition, gene expressions of ASMA, IL-6, MMP-2, TIMP-1 and TIMP-2, all of which are known to be associated with liver fibrosis, were analyzed by using Real-Time PCR. After YBR I and YBR IItreatment, percentages of collagen in TAA-induced rat liver tissue were measured. Results : The viability and proliferation of the HSC-T6 decreased as the concentration increased. The production of procollagen decreased as the concentration increased. The production of IL-6 was little influenced by YBR I and YBR II. There was no difference in rat body weight between the TAA-only group and the YBR groups. Compared with rat liver weight of TAA-only group, that of the YBR groups increased. In the YBR I group, the serum level of AST elevated by TAA injection significantly decreased and in the YBR I and II group, the serum level of ALP and ALT elevated by TAA injection decreased. In the YBR I group, white blood cell count elevated by TAA injection decreased but platelets increased. In the YBR I group, the portal pressure elevated by TAA injection significantly decreased. Decreases in the gene expression of ASMA and MMP-2 were observed in the YBR I group. The gene expression of IL-6 was little influenced by YBR I and YBR II -treated groups. In the histological finding, TAA injections caused severe fibrosis, but YBR I and YBR II treatment significantly reduced the amounts of hepatic collagens. Conclusions : These results suggest that YBR I and II have inhibitory effects on the hepatic fibrogenesis.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Journal of Ginseng Research
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• v.47 no.4
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• pp.515-523
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• 2023

Background: 20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. Methods: The anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. Results: PPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl₄)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. Conclusion: PPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the downregulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

• BMB Reports
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• v.55 no.12
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• pp.633-638
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• 2022

Liver regeneration is a well-known systemic homeostatic phenomenon. The N⁶-methyladenosine (m⁶A) modification pathway has been associated with liver regeneration and hepatocellular carcinoma. m⁶A methyltransferases, such as methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14), are involved in the hepatocyte-specific-regenerative pathway. To illustrate the role of METTL14, secreted from non-parenchymal liver cells, in the initiation phase of liver regeneration, we performed 70% partial hepatectomy (PH) in Mettl14 heterozygous (HET) and wild-type (WT) mice. Next, we analyzed the ratio of liver weight to body weight and the expression of mitogenic stimulators derived from non-parenchymal liver cells. Furthermore, we evaluated the expression of cell cycle-related genes and the hepatocyte proliferation rate via MKI67-immunostaining. During regeneration after PH, the weight ratio was lower in Mettl14 HET mice compared to WT mice. The expressions of hepatocyte growth factor (HGF) and tumor necrosis factor (TNF)-α, mitogens derived from non-parenchymal liver cells that stimulate the cell cycle, as well as the expressions of cyclin B1 and D1, which regulate the cell cycle, and the number of MKI67-positive cells, which indicate proliferative hepatocyte in the late G1-M phase, were significantly reduced in Mettl14 HET mice 72 h after PH. Our findings demonstrate that global Mettl14 mutation may interrupt the homeostasis of liver regeneration after an acute injury like PH by restraining certain mitogens, such as HGF and TNF-α, derived from sinusoidal endothelial cells, stellate cells, and Kupffer cells. These results provide new insights into the role of METTL14 in the clinical treatment strategies of liver disease.

• Preventive Nutrition and Food Science
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• v.18 no.2
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• pp.124-132
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• 2013

In this study, novel peptides (NIPP-1, NIPP-2) derived from Navicula incerta (microalgae) protein hydrolysate were explored for their inhibitory effects on collagen release in hepatic fibrosis with the investigation of its underlying mechanism of action. TGF-${\beta}1$ activated fibrosis in LX-2 cells was examined in the presence or absence of purified peptides NIPP-1 and NIPP-2. Besides the mechanisms of liver cell injury, protective effects of NIPP-1 and NIPP-2 were studied to show the protective mechanism against TGF-${\beta}1$ stimulated fibrogenesis. Our results showed that the core protein of NIPP-1 peptide prevented fibril formation of type I collagen, elevated the MMP level and inhibited TIMP production in a dose-dependent manner. The treatment of NIPP-1 and NIPP-2 on TGF-${\beta}1$ induced LX-2 cells alleviated hepatic fibrosis. Moreover, ${\alpha}$-SMA, TIMPs, collagen and PDGF in the NIPP-1 treated groups were significantly decreased. Therefore, it could be suggested that NIPP-1 has potential to be used in anti-fibrosis treatment.

• Molecules and Cells
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• v.38 no.11
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• pp.998-1006
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• 2015

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis. Con A was injected into wild type (WT), Raldh1 knockout ($Raldh1^{-/-}$), $CCL2^{-/-}$ and $CCR2^{-/-}$ mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-${\gamma}$ in T cells. Moreover, interferon-${\gamma}$ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis. These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.154.1-154.1
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• 2003

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase which plays a crucial role in the synthesis of all collagens, because the 4-hydroxyproline residues are essential for the folding of the newly synthesized collagen polypeptide chains into triple-heical molecules. Considering the role of collagen and its significance in many clinically important diseases such as liver cirrhosis, a great deal of attention has been directed toward the development of an assay at cell-based system. (omitted)

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.300.3-301
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• 2003

The activation of the hepatic stellate cell (HSC) is a key step in liver fibrogenesis. Utilizing large scale sequencing of a 3' -directed cDNA library, we investigated expression profiles of quiescent and activated rat HSCs. During the activation process, O-acetyl disialoganglioside synthase (OAcGD3S) was identified as one of the significant upregulated factors. Upregulation of OAcGD3S in cultured HSCs was confirmed by both northern and western blot analyses. (omitted)

• Proceedings of the Plant Resources Society of Korea Conference
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• 2022.09a
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• pp.106-106
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• 2022

The cone of Red Pine (Pinus densiflora), which has been used as a drug in traditional medicine. Its ethyl acetate fraction was reported to exert antioxidant, anti-melanogenesis, and anti-inflammation activites. Apoptosis of hepatic stellate cells (LX-2) is regarding as a potential strategy for alleviation of hepatic fibrosis. We conducted to investigated whether the treatment of cone has a potential to control of some factors related in apoptosis and autophagy in cell signaling pathways. We suggest that the cone induced apoptosis through confirming the expression levels of genes (cPARP, Bcl-XL, Bax, p53, and caspase-3) in LX-2 cells. Also, the cone may regulate autophagy (LC3, p62, Beclin-1, and ATG12). Remarkably, the treatment of cone may affect to formation of autophagosomes in the immunofluorescence image in live cells. These findings suggest that the ethyl acetate fraction from the cone of Red Pine (P. densiflora) may have potential as an alternative therapeutic agent for the alleviation and prevention of liver fibrosis.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.23 no.4
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• pp.346-355
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• 2020

Purpose: Peroxisome proliferator-activated receptor gamma (PPAR-γ) has a key role in hepatic fibrogenesis by virtue of its effect on the hepatic stellate cells (HSCs). Although many studies have shown that PPAR-γ agonists inhibit liver fibrosis, the mechanism remains largely unclear, especially regarding the cross-talk between PPAR-γ and other potent fibrogenic factors. Methods: This experimental study involved 25 male Wistar rats. Twenty rats were subjected to bile duct ligation (BDL) to induce liver fibrosis, further divided into an untreated group (BDL; n=10) and a group treated with the PPAR-γ agonist thiazolidinedione (TZD), at 14 days post-operation (BDL+TZD; n=10). The remaining 5 rats had a sham operation (sham; n=5). The effect of PPAR-γ agonist on liver fibrosis was evaluated by histopathology, protein immunohistochemistry, and mRNA expression quantitative polymerase chain reaction. Results: Histology and immunostaining showed markedly reduced collagen deposition, bile duct proliferation, and HSCs in the BDL+TZD group compared to those in the BDL group (p<0.001). Similarly, significantly lower mRNA expression of collagen α-1(I), matrix metalloproteinase-2, platelet-derived growth factor (PDGF)-B chain, and connective tissue growth factor (CTGF) were evident in the BDL+TZD group compared to those in the BDL group (p=0.0002, p<0.035, p<0.0001, and p=0.0123 respectively). Moreover, expression of the transforming growth factor beta1 (TGF-β1) was also downregulated in the BDL+TZD group (p=0.0087). Conclusion: The PPAR-γ agonist inhibits HSC activation in vivo and attenuates liver fibrosis through several fibrogenic pathways. Potent fibrogenic factors such as PDGF, CTGF, and TGF-β1 were downregulated by the PPAR-γ agonist. Targeting PPAR-γ activity may be a potential strategy to control liver fibrosis.