• IMMUNE NETWORK
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• v.10 no.6
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• pp.181-187
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• 2010

Excessive alcohol consumption is one of the critical causative factors leading to alcoholic liver disease (ALD). ALD is characterized by a wide spectrum of liver damage, ranging from simple uncomplicated liver steatosis (fatty liver) to steatohepatitis and liver fibrosis/cirrhosis. It has been believed that the obvious underlying cause for ALD is due to hepatocyte death induced by alcohol itself. However, recent sparkling studies have shown that diverse immune responses contribute to ALD because liver is enriched with numerous immune cells. Especially, a line of evidence has suggested that innate immune cells such as Kupffer cells and natural killer (NK)/NKT cells are significantly involved in the pathogenesis of ALD via production of pro-inflammatory cytokines and other mediators. Indeed, more interestingly, hepatic stellate cells (HSCs), known as a major cell inducing liver steatosis and fibrosis, can be killed by liver NK cells, which could be suppressed by chronic alcohol consumption. In this review, with the view of liver as predominant innate immune organ, we describe the pathogenesis of ALD in which what roles of innate immune cells are and how they are interacting with HSCs.

• Archives of Pharmacal Research
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• v.27 no.4
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• pp.422-428
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• 2004

Expressed sequence tags (ESTs) were generated from two 3'-directed CDNA libraries constructed from quiescent and activated rat hepatic stellate cell (HSC) to analyze the expression profiles of active genes in both cells. From quiescent and activated HSC, 694 ESTs and 779 ESTs, respectively, were obtained after excluding those having shorter than 30 bp. Amonq ESTs obtained from quiescent and activated HSC, 68 and 73 kinds of ESTs (186 clones and 236 clones), respectively, appeared more than once, implying that their genes are expressed highly in each cell type. 52 among 73 ESTs appeared only in the activated HSC 47 amonq 68 ESTs only in the normal HSC, and 21 in both cells. The genes of these 52 ESTs were assumed to be expressed more highly in the activated HSC. To confirm the high expression of genes of which the ESTs appeared more than twice in the activated HSC, northern hybridization was carried out with RNAs derived from rat normal and fibrotic liver using each of 18 EST DNAs as probe. 13 ESTs showed more intense bands with RNA isolated from the fibrotic liver than normal liver. From these results, we confirm the positive correlation between abundance of transcript in activated HSCs and the expression level in fibrotic liver, The expression profile of the transcripts serves as an important tool in understanding the biological properties of HSC.

• The Journal of Internal Korean Medicine
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• v.30 no.2
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• pp.306-316
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• 2009

Objectives : This study was performed to investigate the anti-fibrogenic effect of Curcumae Longae Radix on human hepatic stellate cells. Materials and Methods: Hepatic stellate cells (LX-2) were treated with various concentrations of Curcumae Longae Radix extract for 24, 48, and 72 hours. It was extracted with distilled water. After the treatment, cell viability, proliferation, cell cycle analysis, procollagen levels and the mRNA of the ASMA, TIMPl, TIMP2, MMP2, collagen type la, PDGF-receptor-beta and TGF-beta were measured by using MTT assay, BrdU assay, RT-PCR, and procollagen type 1 C-peptide EIA kit. Results : The viability of HSCs decreased in the 48 hours group, and proliferation of HSCs decreased as the concentration increased. In the cell cycle analysis, Curcumae Longae Radix decreased the ratio of M phase, and increased the ratio of apoptosis, G0/G1 and S phase. In the RT-PCR, the mRNA expression of the collagen type la and ASMA decreased with the Curcumae Longae Radix treatment. The production of procollagen by the HSCs was decreased by the treatment of Curcumae Longae Radix with high dose. Conclusion : These results suggest that Curcumae Longae Radix is helpful in the treatment of liver fibrosis as well as liver cirrhosis.

• BMB Reports
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• v.53 no.8
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• pp.425-430
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• 2020

Tumor necrosis factor-inducible gene 6 protein (TSG-6) is a cytokine secreted by mesenchymal stem cells (MSCs) and regulates MSC stemness. We previously reported that TSG-6 changes primary human hepatic stellate cells (pHSCs) into stem-like cells by activating yes-associated protein-1 (YAP-1). However, the molecular mechanism behind the reprogramming action of TSG-6 in pHSCs remains unknown. Cluster of differentiation 44 (CD44) is a transmembrane protein that has multiple functions depending on the ligand it is binding, and it is involved in various signaling pathways, including the Wnt/β-catenin pathway. Given that β-catenin influences stemness and acts downstream of CD44, we hypothesized that TSG-6 interacts with the CD44 receptor and stimulates β-catenin to activate YAP-1 during TSG-6-mediated transdifferentiation of HSCs. Immunoprecipitation assays showed the interaction of TSG-6 with CD44, and immunofluorescence staining analyses revealed the colocalization of TSG-6 and CD44 at the plasma membrane of TSG-6-treated pHSCs. In addition, TSG-6 treatment upregulated the inactive form of phosphorylated glycogen synthase kinase (GSK)-3β, which is a negative regulator of β-catenin, and promoted nuclear accumulation of active/nonphosphorylated β-catenin, eventually leading to the activation of YAP-1. However, CD44 suppression in pHSCs following CD44 siRNA treatment blocked the activation of β-catenin and YAP-1, which inhibited the transition of TSG-6-treated HSCs into stem-like cells. Therefore, these findings demonstrate that TSG-6 interacts with CD44 and activates β-catenin and YAP-1 during the conversion of TSG-6-treated pHSCs into stem-like cells, suggesting that this novel pathway is an effective therapeutic target for controlling liver disease.

• The Korea Journal of Herbology
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• v.28 no.4
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• pp.49-55
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• 2013

Objectives : Mori Folium was popularly used as one of the traditional medicinal herbs. Although M. Folium has been cultivated for rearing silkworm historically, it's use has been expanded as natural therapeutic agent for the treatment of filariasis, diabetes and dropsy in East Asia. However, little has been known about the effect of M. Folium on liver fibrosis. Therefore, we would like to explore an anti-fibrogenic potential of M. Folium extract (MFE) using immortalized human hepatic stellate cell line, LX-2 cells. Methods : We examined the effects of MFE on the transforming growth factor ${\beta}1$ ($TGF{\beta}1$)-induced liver fibrosis in LX-2 cells. Cell viability, Smad binding element-driven luciferase activity, phosphorylations level of Smad 2/3, and expression level of $TGF{\beta}1$-dependent target genes were monitored in the MFE-treated LX-2 cells. Results : Up to 30 ${\mu}g/ml$ MFE treatment did not show any possible toxic effect in LX-2 cells. MFE inhibited $TGF{\beta}1$-inducible Smad binding element-driven luciferase activity and decreased the $TGF{\beta}1$-inducible phosphorylations of Smad 2 and Smad 3 in hepatic stellate cell in a dose dependent manner. Furthermore, increases of plasminogen activator inhibitor type 1, $TGF{\beta}1$ and matrix metalloproteinases 2 genes by $TGF{\beta}1$ were also attenuated by MFE treatment. Conclusions : These findings suggested that MFE would be used as a potential therapeutic agent for the treatment liver fibrosis, which might be mediated by the inhibition of $TGF{\beta}1$-inducible Smad 2/3 transactivation and target genes expression.

• 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.

• Herbal Formula Science
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• v.26 no.3
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• pp.183-193
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• 2018

Objectives : In Traditional Korean Medicine, Epimedium koreanum Nakai has diverse pharmacological activities to treat impotence, forgetfulness, cataract and exophthalmos. Present study investigated anti-fibrogenic effects of E. koreanum water extract (EKE) in hepatic stellate cells (HSCs). Methods : To study anti-fibrogenic effects of EKE, LX-2 cells, a human immortalized HSCs, were pre-treated with $3-300{\mu}g/mL$ of EKE, and then subsequently exposed to 5 ng/mL of transforming growth $factor-{\beta}1$ ($TGF-{\beta}1$). Expression level of ${\alpha}-smooth$ muscle actin was determined by immunoblot analysis. Phosphorylation of Smad, transactivation of Smad, and expression of plasminogen activator inhibitor-1 (PAI-1) were monitored to investigate the effect of EKE on $TGF-{\beta}1-mediated$ signaling pathway. Results : Up to $100{\mu}g/mL$, EKE did not show any cytotoxicity on LX-2 cells. Pre-treatment of EKE ($100{\mu}g/mL$) significantly inhibited ${\alpha}-smooth$ muscle actin expression induced by $TGF-{\beta}1$. In addition, EKE significantly decreased Smad2 and Smad3 phosphorylations, Smad binding element-driven luciferase activity and PAI-1 expression by $TGF-{\beta}1$. Of three flavonoid compounds found in EKE, only quercertin ($30{\mu}M$) attenuated $TGF-{\beta}1-mediated$ PAI-1 expression. Conclusion : These results suggest that EKE has an ability to suppress fibrogenic process in HSCs via inhibition of $TGF-{\beta}1/Smad$ signaling pathway.

• Advances in Traditional Medicine
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• v.10 no.4
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• pp.254-262
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• 2010

Artemisia capillaris (A. capillaries) is known to play roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We investigated the antifibrogenic efficacy of A. capillaris in the immortalized human hepatic stellate cell line LX2. Cell proliferation was determined by the MTT assay. Cell cycle was analyzed by the flow cytometry. Apoptotic cells were measured using a cell death detection ELISA. Caspase activity was detected by a colorimetric assay. The mRNA level of Bcl-2 and Bax mRNA were measured by real-time PCR. MEK and ERK protein were detected by Western blot analysis. We provide evidence that A. capillaris induces cell cycle arrest, apoptosis, and potently inhibits the mitogen-activated protein kinase pathway. A. capillaris inhibited cell proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle analysis with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 mRNA levels and an increase in Bax expression. Exposure of LX2 cells to A. capillaris induced caspase-3 activation, but co-treatment of A. capillaris with the pan-caspase inhibitor Z-VAD-FMK, and the caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. A. capillaris down-regulated Mcl-1 protein levels and inhibited phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.

• Molecules and Cells
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• v.25 no.3
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• pp.376-384
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• 2008

The glial fibrillary acidic protein (GFAP) is traditionally used as a marker for astrocytes of the brain, and more recently for the hepatic stellate cells (HSCs) of the liver. Several GFAP splice variants have been previously reported in the astrocytes of the CNS and in the non-myelinating Schwann cells of the PNS. In this study, we investigate whether GFAP splice variants are present in the HSCs and their expression as a function of HSCs activation. Furthermore, the regulation of these transcripts upon treatment with interferon gamma ($IFN-{\gamma}$) will be explored. Using semi-quan-titative RT-PCR and real-time PCR, we examine the expression and regulation of GFAP splice variants in HSCs as well as their respective half-life. We discover that most of the GFAP splice variants ($GFAP{\alpha}$, ${\beta}$, ${\delta}$, ${\varepsilon}$ and $\kappa$) found in the neural system are also expressed in quiescent and culture-activated primary HSCs. Interestingly, $GFAP{\alpha}$ is the predominant form in quiescent and culture-activated primary HSCs, while $GFAP{\beta}$, predominates in the SV40-immortalized activated HSC-T6. $GFAP{\delta}$, ${\varepsilon}$ and ${\kappa}$ have similar half-lives of 10 hours, while $GFAP{\beta}$ has a half-life of 17 hours. Treatment of HSC-T6 with $IFN-{\gamma}$ results in a significant 1.29-fold up-regulation of $GFAP{\alpha}$ whereas the level of the other transcripts remains unchanged. In summary, $GFAP{\alpha}$, ${\beta}$, ${\delta}$, ${\varepsilon}$ and $\kappa$ are present in HSCs. They are differentially regulated on the transcription level, implying a role of the 5' and 3' untranslated regions.

• Biomolecules & Therapeutics
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• v.15 no.4
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.