• 대한한방내과학회지
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• 제30권2호
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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.

• 대한한방내과학회지
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• 제31권3호
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• pp.415-424
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• 2010

Objectives : This study was performed to investigate the anti-fibrogenic effect of Angelica Gigantis Radix on cultured rat hepatic stellate cells. Materials and Methods : Hepatic stellate cells(HSC-T6) were treated with various concentrations of Angelica Gigantis Radix extract for both 24 and 48 hours. The extraction was done either with distilled water or 80% EtOH. After the treatment, cell viability, cell proliferation, procollagen production and the mRNA expression of the ASMA, TIMP1, TIMP2, procollagen Type 1a2, and Cytokine IL-6 production were measured by using MTT assay, BrdU assay, RT-PCR, procollagen Type I C-peptide EIA and IL-6 ELISA assay. Results : The cell viability treated with water extraction was significantly increased, but there were no significant changes treated with 80% EtOH extraction. The cell proliferation treated with water extraction decreased only in the 24 hours group, while there were significant decreases either in the 24 and 48 hours groups treated with 80% EtOH extraction. The mRNA expressions of the ASMA, TIMP2 and procollagen 1a2 decreased in a concentration-dependent manner in the 48 hours group. Procollagen production decreased in a concentration-dependent manner in both the 24 and 48 hours groups. Cytokine IL-6 production increased in a concentration-dependent manner in both the 24 and 48 hours groups. Conclusion : These results suggest that Angelica Gigantis Radix is beneficial in the treatment of cirrhotic patients as well as for patients with chronic hepatitis.

• 대한한방내과학회지
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• 제31권2호
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• pp.346-355
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• 2010

Objectives : This study was performed in order to investigate the anti-fibrogenic effect of Acer tegmentosum Maxim. on r at hepatic stellate cell line T6. Materials and Methods : Hepatic stellate Cells (T6) were treated with various concentrations of distilled water Acer teg mentosum Maxim. extract for 24, 48, 72 hours. After the treatment, cell viability, proliferation, procollagen levels, mRNA of AS MA, MMP-2, collagen type 1a2 and IL-6 production were measured using MTT assay, BrdU assay, RT-PCR, procollagen typ e 1 C-peptide EIA kit and murine IL-6 ELISA development kit. Results : Cell viability of HSC-T6 decreased significantly in both 24 hours and 48 hours groups in a dose-dependant man ner. Proliferation of HSC also decreased in the same way. In the RT-PCR, mRNA expression of collagen type 1a2 and ASMA decreased in the groups which were treated with Acer tegmentosum Maxim. for 24 hours. The production of procollagen tended to decrease in a dose-dependant manner in the 24 hours treated group. IL-6 production increased under Acer tegmentosum trea tment in a dose-dependant manner in both 24 and 48 hours groups. Conclusion : These results show the possibility that Acer tegmentosum Maxim. can be an effective remedy for liver fibrosi s and liver cirrhosis.

• Journal of Applied Biological Chemistry
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• 제66권
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• pp.138-143
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• 2023

Liver fibrosis is caused by metabolic problems such as cholestasis, genetic problems, or viral infections. Inhibiting hepatic stellate cell (HSC) activation or inducing selective apoptosis of activated HSCs is used as a treatment strategy for liver fibrosis. It has been reported that when HSCs are activated, their apoptosis sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is enhanced because the expression of death receptor 5 is elevated. Finding a natural compound that can enhance the apoptotic effect of TRAIL on HSCs is a necessary strategy for liver fibrosis treatment. It was confirmed here that mangosteen-derived gartanin increased the effect of TRAIL-induced apoptosis by increasing the expression of DR5 in a p38-dependent manner in the hepatic stellate cell line LX-2. Combined treatment with gartanin and TRAIL accelerated DNA cleavage through caspase-3 activation and enhanced antifibrotic effects in LX-2 cells.

• Archives of Pharmacal Research
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• 제27권4호
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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.

• Advances in Traditional Medicine
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• 제10권4호
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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.

• Biomolecules & Therapeutics
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• 제15권4호
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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.

• 대한한방내과학회지
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• 제26권4호
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• pp.853-863
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• 2005

Object : This study was performed to investigate the anti-fibrogenic effect of Artemisiae Capillaris Herba(ACH) on cultured rat hepatic stellate cells. Methods : Hepatic Stellate Cells were obtained from a 350gm Sprague-Dawley rat by tissue perfusion system, and the cells for the study were selected after 3 passages of culture on non-coated plastic culture dishes which enable the cells to activate, thus producing collagens in the cell media. Cells were treated with various concentrations of Artemisia Capillaris Herba(ACH) extract powder for 24 or 48 hours. After the treatment, Soluble collagen, procollagen levels and the mRNA of the procollagen type I C were measured by using assay kit and RT-PCR method. Results : Procollagen production by the hepatic stellate cells decreased after the treatment in a time-dependent dose-dependent manner. The mRNA expression decreased consistently with the volume of the secreted procollagen which indicates the herb hat inhibitory effect on fibrogenesis of the liver by regulating one of the fibrosis associated genes in transcription. Conclusion : These results suggest that ACH is beneficial in the treatment of cirrhotic patients as well as for the patients with chronic hepatitis.

• 대한한방내과학회지
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• 제29권2호
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• pp.299-310
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• 2008

Objectives : This study was performed to investigate the anti-fibrogenic effect of Salvia miltiorrhiza on rat hepatic stellate cells. Materials and Methods : Hepatic stellate cells(HSC-T6) were treated with various concentrations of Salvia miltiorrhiza extract for 24 hours. It was extracted either with distilled water or 50% EtOH. After the treatment, cell viability, proliferation, procollagen levels and the mRNA of the collagen type 1a2 and ASMA were measured Results : The viability and proliferation of the hepatic stellate cells were decreased as concentration increased. The mRNA expression decreased consistently with the volume of the secreted procollagen with the extraction made with distilled water. This indicates the herb has inhibitory effect on fibrogenesis of the liver by regulating one of the fibrosis associated genes in transcription. However it increased in 50% EtOH extraction, which shows that a more stable reaction is expected of the extraction made with distilled water than the extraction made with 50% EtOH. The production of procollagen was decreased by a low-concentration treatment with Salvia miltiorrhiza, but increased by a high concentration. It seemed that the cells were responding to Salvia miltiorrhiza in low- concentration, thus producing smaller amounts of collagen. When the drug was administered in high enough concentration to give direct toxicity to cells, an overproduction of collagen was observed. Conclusion : These results suggest that Salvia miltiorrhiza is a possible candidate for the treatment of cirrhotic patients as well as for patients with chronic hepatitis when extracted with water in the proper concentrations.

• 대한한방내과학회지
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• 제29권1호
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• pp.177-188
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• 2008

Objectives : This study was performed to investigate the anti-fibrogenic effect of Artemisiae Capillaris Herba on cultured rat hepatic stellate cells. Materials and Methods : Hepatic stellate cells(HSC-T6) were treated with various concentrations of Artemisiae Capillaris Herba extract for 24 hours. The extraction was done either with distilled water or 50% EtOH. After the treatment, cell viability, proliferation, procollagen levels and the mRNA of the collagen type 1a2 and ASMA were measured by using MTT assay, BrdU assay, RT-PCR, and Procollagen Type I C-peptide EIA Kit. Results : The viability and proliferation of the hepatic stellate cells were decreased as the concentration increased. The mRNA expression decreased consistently with the volume of the secreted procollagen with the extraction made with distilled water, which indicates the herb has inhibitory effect on fibrogenesis of the liver by regulating one of the fibrosis associated genes in transcription. However, it increased in 50% EtOH extraction, which shows that a more stable reaction is expected of the extraction made with distilled water than the extraction made with 50% EtOH. The production of procollagen was decreased by a low-concentration treatment with Artemisiae Capillaris Herba, but increased by a high concentration. It seemed that the cells were responding to Artemisiae Capillaris Herba in low- concentrations, thus producing small amounts of collagen. When the drug was administered at high enough concentration to give direct toxicity to cells, the ability of cells to produce collagen was activated, and the overproduction of collagen was observed as an undesirable results. Conclusion : These results suggest that Artemisiae Capillaris Herba is beneficial in the treatment of cirrhotic patients as well as for the patients with chronic hepatitis when extracted with water in the proper concentrations.