• 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.

• Archives of Pharmacal Research
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• 제29권9호
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• pp.762-767
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• 2006

Danshen is an herbal medication frequently used in oriental medicine to treat liver or kidney malfunction. In the course of our studies, we observed that compounds purified from Danshen exhibit an inhibitory activity against Discoidin Domain Receptor 2 (DDR2) tyrosine kinase. Through this inhibition, these compounds also inhibited the growth of HSC T6 cells and suppressed the expression of alpha-smooth muscle actin and MMP2, as well as collagen synthesis, all of which are increased in activated liver stellate cells. Given that activation of liver stellate cells is the hallmark of liver fibrosis and that DDR2 plays a critical role in this activation, these results suggest that one of the pharmacological activities of Danshen extract that protects the liver is the inhibition of key cell-signaling kinases, such as DDR2, in liver stellate cells.

• Nutrition Research and Practice
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• 제4권5호
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• pp.369-374
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• 2010

Grape is one of the most popular and widely cultivated fruits in the world. Although grape skin and seeds are waste product of the winery and grape juice industry, these wastes contain large amounts of phytochemicals such as flavonoids, phenolic acids, and anthocyanidins, which play an important role as chemopreventive and anticancer agents. We evaluated efficacies of grape skin and seeds on hepatic injury induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN significantly increased levels of serum alanine transaminase, aspartate transaminase, alkaline phosphatase, and bilirubin. Diet supplementation with grape skin or seeds (10% daily for 4 weeks) prevented these elevations. The grape skin and seeds also restored serum albumin and total protein levels, and reduced the hepatic level of hydroxyproline and malondialdehyde. Furthermore, grape skin and seeds reduced DMN-induced collagen accumulation, as estimated by histological analysis of liver tissue stained with Sirius red. Grape skin and seeds also reduced hepatic stellate cell activation, as assessed by ${\alpha}$-smooth muscle actin staining. In conclusion, grape skin and seeds exhibited in vivo hepatoprotective and antifibrogenic effects against DMN-induced liver injury, suggesting that grape skin and seeds may be useful in preventing the development of hepatic fibrosis.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2003년도 추계학술대회
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• pp.171-171
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• 2003

Asiaticoside, a biologically active triterpenoid present in Centella asicatica, has been known to exert a variety of biological effect such as wound-healing, hepatoprotective, anti-inflammatory. In this study, we observed the effect of asiatic acid asiaticoside and its 16 derivatives on the cytotoxicity and the content of hydroxyproline in rat hepatic stellate cell line, HSC-T6 cell as a preliminary study for screening the anti-hepatofibrotic effect.(omitted)

• IMMUNE NETWORK
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• 제10권6호
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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.

• Natural Product Sciences
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• 제14권2호
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• pp.118-121
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• 2008

Manassantin B, a dilignan isolated from Saururus chinensis, significantly inhibited proliferation in HSC-T6 cells in concentration- and time-dependent manners. In addition, treatment of HSC-T6 cells with manassantin B changed cell morphology from flattened myofibroblastic membranous morphology, representing activation state, to slender shape, representing quiescent state. Furthermore, manassantin B effectively reduced collagen content in HSC-T6 cells. These results suggested that manassantin B exerted antifibrotic activity in HSCT6 cells, in part, via inhibition of cell proliferation and decrease of collagen production.

• 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.

• Preventive Nutrition and Food Science
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• 제18권2호
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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.

• 대한본초학회지
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• 제28권4호
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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.

• Biomolecules & Therapeutics
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• 제30권2호
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• pp.162-169
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• 2022

We utilized Fas21, a resveratrol analog, to modulate the function of hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) during the angiogenic phase of murine liver metastasis by B16 melanoma and 51b colorectal carcinoma. Preangiogenic micrometastases were treated with Fas21 (1 mg/kg/day) or vehicle during the development of intra-angiogenic tracts. Mice treated with Fas21 showed reduced liver tumor foci in both liver metastasis models. Micrometastases were classified immunohistochemically, as well as according to their position coordinates and connection to local microvasculature. The volume of liver occupied by sinusoidal-type foci, containing infiltrating angiogenic capillaries, decreased by ~50% in Fas21-treated mice compared to vehicle-treated ones in both tumor metastasis models. The volume of portal foci, containing peripheral neoangiogenesis within a discontinuous layer of myofibroblasts, was similar in all experimental groups in both tumor metastasis models, but displayed enhanced necrotic central areas devoid of angiogenesis following Fas21 treatment. As a result, sinusoidal tumors from mice treated with Fas21 showed a 50% reduction in desmin(+)/asma(+) HSCs and CD31(+) vessel density, and a 45% reduction in intrametastatic VEGF mRNA compared with sinusoidal tumors from vehicle-treated mice. Necrotic portal metastases increased 2-4-fold in treated mice. In vitro, Fas21 reduced VEGF secretion by HSCs and 51b cells dose-dependently. Additionally, HSCs migration in response to tumor soluble factors was dose-dependently diminished by Fas21, as was LSEC migration in response to HSCs and tumor soluble factors. Resveratrol analog Fas21 inhibits the proangiogenic response of HSCs and LSECs during the development of murine liver metastasis.