• 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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• 제31권4호
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• pp.425-433
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• 2023

During liver injury, hepatic stellate cells can differentiate into myofibroblast-like structures, which are more susceptible to proliferation, migration, and extracellular matrix generation, leading to liver fibrosis. Anaerobic glycolysis is associated with activated stellate cells and glyceraldehyde (GA) is an inhibitor of glucose metabolism. Therefore, this study aimed to investigate the anti-fibrotic effects of GA in human stellate LX-2 cells. In this study, we used cell viability, morphological analysis, fluorescence-activated cell sorting (FACS), western blotting, and qRT-PCR techniques to elucidate the molecular mechanism underlying the anti-fibrotic effects of GA in LX-2 cells. The results showed that GA significantly reduced cell density and inhibited cell proliferation and lactate levels in LX-2 cells but not in Hep-G2 cells. We found that GA prominently increased the activation of caspase-3/9 for apoptosis induction, and a pan-caspase inhibitor, Z-VAD-fmk, attenuated the cell death and apoptosis effects of GA, suggesting caspase-dependent cell death. Moreover, GA strongly elevated reactive oxygen species (ROS) production and notably increased the phosphorylation of ERK and JNK. Interestingly, it dramatically reduced α-SMA and collagen type I protein and mRNA expression levels in LX-2 cells. Thus, inhibition of ERK and JNK activation significantly rescued GA-induced cell growth suppression and apoptosis in LX-2 cells. Collectively, the current study provides important information demonstrating the anti-fibrotic effects of GA, a glycolytic metabolite, and demonstrates the therapeutic potency of metabolic factors in liver fibrosis.

• Toxicological Research
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• 제18권4호
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• pp.363-367
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• 2002

Reactive of gen species (ROS) contribute to several cellular function and are involved in the regulation of signal transduction, gene expression, and proliferation. In the present study, we investigated the effect of $H_2O_2$ treatment on IgM secretion in LPS-stimulated murine B Iymphoma, CH12.LX. Cells were treated directly With $H_2O_2$ and stimulated with LPS. $H_2O_2$ treatment during 72 h time period inhibited IgM secretion in LPS-stimulated CH12.LX cells in a dose- and time-dependent manners. After treatment with 50 $\mu\textrm{M}$ $H_2O_2$ during 72 h time period, the level of IgM in LPS-stimulated CH12.LX cells was markedly decreased, whereas cell viability was not significantly changed. Addition of $H_2O_2$ concomitantly with LPS, or 12 h post-LPS stimulation, produced a significant inhibition of IgM secretion, Whereas inhibitory effect of $H_2O_2$ on IgM secretion was not observed when added 24 h after LPS stimulation. These findings suggest that $H_2O_2$ can inhibit the secretion of IgM in LPS-stimulated CH15.LX cells, and may alter the events necessary for terminal B cell differentiation.

• 대한본초학회지
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• 제39권4호
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• pp.19-28
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• 2024

Objectives : Ephedrae Herba has been used in the East Asian traditional medicine, for treatment of asthma, cold and influenza. Silymarin is an effective antioxidant and its anti-fibrogenic, anti-inflammatory, and hepatoprotective properties have been reported. This study was performed to explore an anti-fibrogenic potential of Ephedrae Herba extract (EHE) + silymarin on immortalized human hepatic stellate cell line, LX-2 cells. Methods : We studied the anti-fibrogenic effects of EHE + silymarin on transforming growth factor β1 (TGF-β1) signaling pathway in LX-2 cells. Cell viability was measured using the MTT assay. mRNA levels were detected by real-time PCR. TGF-β1 signaling-related proteins expression were detected by Western blot. Results : Silymarin 30 ㎍/mL and EHE 100 ㎍/mL showed cytotoxicity on LX-2 cells. Therefore, the concentrations of silymarin and EHE were studied at 10 ㎍/mL, respectively. Silymarin significantly reduced PAI-1 protein expression, Smad binding element (SBE) luciferase activity, and mRNA (PAI-1, MMP2 and 9) expression compared to TGF-β1. EHE significantly reduced SBE luciferase activity and mRNA (PAI-1, MMP2 and 9) expression compared to TGF-β1. More importantly, EHE + silymarin significantly reduced all parameters compared to TGF-β1, and also significantly reduced compared to EHE alone and silymarin alone. Conclusion : The results indicate that EHE + silymarin has anti-fibrogenic effect in LX-2 cells induced by TGF-β1. Additionally, EHE + silymarin shows more effective anti-fibrogenic effect than EHE alone and silymarin alone.

• 대한한방내과학회지
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• 제32권4호
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• pp.519-529
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• 2011

Objectives : This study was performed to investigate the effects of Injinchunggan-tang on cell growth and apoptosis in human hepatic stellate cell line LX2. Materials and Methods : Hepatic stellate cells were treated with various concentrations of Injinchunggan-tang extract for 24, 48 and 72 hours. The extraction was done with distilled water. After the treatment, cell viability, proliferation, apoptosis, caspase activity, caspase inhibitor and the mRNA of the Bcl-2, and Bax with ${\beta}$-actin were measured by using MTT assay, apoptosis assay and RT-PCR. Results : Proliferation, and mRNA expression of the hepatic stellate cells were inhibited by Injinchunggan-tang treatment in a dose-dependent manner. This indicates the prescription has inhibitory effect on fibrogenesis of the liver by regulating the fibrogenesis-associated genes in transcription. Cell viability was inhibited in time- and dose-dependent manners. Conclusions : These results suggest that Injinchunggan-tang would be beneficial in the treatment of cirrhotic patients as well as for the patients with chronic hepatitis.

• 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.

• 대한한의학방제학회지
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• 제32권2호
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• pp.141-153
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• 2024

Objective : Ephedrae Herba (Ephedra) has been frequently used in the East Asian traditional medicine including Korea, China and Japan in the clinical treatment of asthma, cold and influenza etc. This study was performed to explore an anti-fibrogenic potential of Ephedra Herba water extract (EHE) using immortalized human hepatic stellate cell line, LX-2 cells. Methods : We examined the anti-fibrogenic effects of EHE on canonical pathway of transforming growth factor-β1 (TGF-β1) signaling in LX-2 cells. Cell viability was measured using the MTT assay. mRNA levels were detected by real-time PCR. Proteins expression were detected by Western blot. Results : Treatment of EHE 30 ㎍/ml did not show any cytotoxicity on LX-2 cells. Pre-treatment of EHE (30 ㎍/mL) significantly inhibited α-smooth muscle actin expression induced by TGF-β1. Additionally, EHE significantly decreased Smad2 and Smad3 phosphorylations, Smad binding element-driven luciferase activity and plasminogen activator inhibitor type 1 expression by TGF-β1. Furthermore, increases of matrix metalloproteinases 2 genes by TGF-β1 was also attenuated by EHE treatment. Conclusion : These results suggest that EHE has an ability to suppress fibrogenic process in activated HSC via inhibition of TGF-β1-TGFBR mediated canonical (Smad dependent) pathway.

• 대한한방내과학회지
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• 제32권2호
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• pp.153-169
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• 2011

Objectives : This study was performed to investigate the anti-fibrogenic effect and the effect on cell growth and apoptosis in YJCGT, YJCGT YSO and YJCGT YSCO on thioacetamide-induced rat liver tissue and the immortalized human hepatic cell line LX2. Materials and Methods : LX2 cells were treated with various concentrations (0, 50, 150, 300 ug/ml) of YJCGT, Y+YSO, and Y+YSCO extract for 24, 48 and 72 hours. After the treatment, cell viability was measured by using MTT assay. Caspase inhibitor assay, and cell viability were determined by a colorimetric assay with PMS/MTS solution. Rat liver fibrosis was induced by intraperitoneal thioacetamide injection 150 mg/kg 3 times a week for 5 weeks. After the treatment, body weight, liver & spleen weights, liver function test, the complete blood cell count and the change of portal pressure were studied. After YJCGT, Y+YSO, and Y+YSCO treatment, percentages of collagen in thioacetamide-induced rat liver tissue were measured. Results : The viability of the LX2 cell decreased in a dose- and time-dependent manner. Exposure of LX2 cells to YJCGT, YJCGT+YSO and YJCGT+YSCO induced caspase-3 activation, but co-treatment of YJCGT, YJCGT+YSO and YJCGT+YSCO with the pan-caspase inhibitor Z-VAD-FMK, and the caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. There was no difference in rat body weight between the thioacetamide only group and the YJCGT, YJCGT+YSO and YJCGT+YSCO groups. In the YJCGT, YJCGT YSO and YJCGT YSCO groups, the serum level of GPT significantly went down compared with the thioacetamide only group. In the YJCGT, Y+YSO, Y+YSCO groups, white blood cell elevated by thioacetamide injection decreased but RBC, Hgb, and Hct increased. In the Y+YSO group, the portal pressure elevated by thioacetamide injection significantly decreased. In the histological finding, thioacetamide injections caused severe fibrosis, but YJCGT, Y+YSO, and Y+YSCO treatment significantly reduced the amounts of hepatic collagens. Conclusions : YJCGT, Y+YSO, and Y+YSCO inhibit the growth of LX2 cells by inducing apoptosis through caspase activity. YJCGT, Y+YSO, and Y+YSCO have beneficial effects on the treatment of cirrhotic patients as well as patients with chronic hepatitis.

• 미생물학회지
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• 제23권1호
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• pp.20-24
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• 1985

Klebsiella pnellmoniae의 nif-lac융합변이주를 사용하여 질소고정 유전자 발현에 미치는 질소원의 효과를 검토하였다. Klebsiella pnellmoniae UN4482를 heat induction하여 만든 Mudllysate를 K. pnellmoniae U UN2979에 접종시켜 nif-lac융합체 약 80여주를 분리하고 이들을 LX series로 명하였다. 이들의 ${\beta}-galactosidase$ 한성은 $NH_4^+$의 존재하에 크게 억제되었다. Serine, glutamine, asparagine같은 아미노산을 켈소원으로 사용했을때 K. pnellmoniae의 성장은 양호하였으며. NFHM배지에서nif-lac융합주LX-9, LX-22의 ${\beta}-galactosidase$의 환성은 억제 효과도 높았으나, glutamine, histidine, arginine 같은 amino acid는 위와 반대 의 효과플 나타냈다. Casitone, prote-ose peptone같은 유기섣소원(2 mg/ml )의 균성장에 대한효과는 전반적으로양호했으나LX-9, LX-22 의 ${\beta}-galactosidase$활성에 대한 억제효과는 각 칠소원에 따라 다르게 나타났다. 한편, 질소원이 없는 최소배지에서의 LX-9와 L LX-22의 ${\beta}-galactosidase$의 활성은 초기 4 시간내에 급격히 증가하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7629-7634
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• 2013

Background: Cylin D1(CCDN1) is an important regulator of the cell cycle whose alterations are thought to be involved in cancer development. There have been many studies indicating CCDN1 amplification or over-expression in a variety of cancer types. In addition to gene amplification, the G870A polymorphism may be related with altered CCDN1 activity, and therefore with cancer development. This hypothesis has been tested in different cancer types but results have been contradictory. We therefore aimed to investigate any relationship between CCDN1 A870G genotypes and laryngeal squamous cell cancer development and progression. Materials and Methods: A total of 68 Turkish patients with primary laryngeal squamous cell cancer and 133 healthy controls were enrolled. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to determine the CCDN1 genotypes. Results: No significant association was detected between CCDN1 genotypes and laryngeal squamous cell cancer (LxSCCa) development. Similarly CCDN1 genotypes were not related to clinical parameters of Lx SCCa. However, there was a very significant association between CCDN1 G allele and presence of perineural invasion (p=0.003; OR: 1.464; CI% 1.073-1.999). CCDN1 G allele frequency was significantly higher in the individuals with perineural invasion (85.7%) when compared to those without (58.5%). The 2 patients who died of disease were both found to possess the GG genotype. Conclusions: These results pose a controversy in suggesting a protective role of the G allele against LxSCCa development and support the association of CCDN1 gene GG genotype with mortality in patients with LxSCCa.