The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

1992 ◽  
Vol 68 (01) ◽  
pp. 040-047 ◽  
Author(s):  
C Scott Jamison ◽  
Bryan F Burkey ◽  
Sandra J Friezner Degen
Keyword(s):  
Total Protein ◽  
Hepg2 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Analysis ◽  
Secreted Protein ◽  
Mrna Levels ◽  
Vitamin K1 ◽  
Maximal Increase ◽  
Protein Levels ◽  
Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

Abstract TP274: Orosomucoid-1 Protein Increases Following Ischemic Stroke in the Brain and Periphery

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Hetal Mistry ◽  
Madeline Levy ◽  
Meaghan Roy-O'Reilly ◽  
Louise McCullough
Keyword(s):  
Sex Differences ◽  
Ischemic Stroke ◽  
Rna Sequencing ◽  
Multiple Testing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Stroke Patients ◽  
Post Stroke ◽  
Protein Levels ◽  
The Brain

Background and Purpose: Orosomucoid-1 (ORM-1) is an abundant protein with important roles in inflammation and immunosuppression. We utilized RNA sequencing to measure mRNA levels in human ischemic stroke patients, with confirmation by serum ORM-1 protein measurements. A mouse model of ischemic stroke was then used to examine post-stroke changes in ORM-1 within the brain itself. Hypothesis: We tested the hypothesis that ORM-1 levels increase following ischemic stroke, with sex differences in protein dynamics over time. Methods: RNA sequencing was performed on whole blood from ischemic stroke patients (n=23) and controls (n=12), with Benjamini-Hochberg correction for multiple testing. Enzyme-linked immunosorbent assay was performed on serum from ischemic stroke patients (n=28) and controls (n=8), with analysis by T-test. For brain analysis, mice (n=14) were subjected to a 90-minute middle cerebral artery occlusion (MCAO) surgery and sacrificed 6 or 24 hours after stroke. Control mice underwent parallel “sham” surgery without occlusion. Western blotting was used to detect ORM-1 protein levels in whole brain, with analysis by two-way ANOVA. Results: RNA sequencing showed a 2.8-fold increase in human ORM-1 at 24 hours post-stroke (q=.0029), an increase also seen in serum ORM-1 protein levels (p=.011). Western blot analysis of mouse brain revealed that glycosylated (p=0.0003) and naive (p=0.0333) forms of ORM-1 were higher in female mice compared to males 6 hours post-stroke. Interestingly, ORM-1 levels were higher in the brains of stroke mice at 6 hours (p=.0483), while at 24 hours ORM-1 levels in stroke mice were lower than their sham counterparts (p=.0212). In both human and mouse data, no sex differences were seen in ORM-1 levels in the brain or periphery at 24 hours post-stroke. Conclusion: In conclusion, ORM-1 is a sexually dimorphic protein involved in the early (<24 hour) response to ischemic stroke. This research serves as an initial step in determining the mechanism of ORM-1 in the ischemic stroke response and its potential as a future therapeutic target for both sexes.

scholarly journals Activation of Nrf2 by Electrophiles Is Largely Independent of the Selenium Status of HepG2 Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 167
Author(s):  
Sarah Tauber ◽  
Maria Katharina Sieckmann ◽  
Katrin Erler ◽  
Wilhelm Stahl ◽  
Lars-Oliver Klotz ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Time Course ◽  
Target Genes ◽  
Intracellular Localization ◽  
Redox Homeostasis ◽  
Major Effect ◽  
Related Factor ◽  
Mrna Levels ◽  
Protein Levels ◽  
Se Status

Selenoenzymes, whose activity depends on adequate selenium (Se) supply, and phase II enzymes, encoded by target genes of nuclear factor erythroid 2-related factor 2 (Nrf2), take part in governing cellular redox homeostasis. Their interplay is still not entirely understood. Here, we exposed HepG2 hepatoma cells cultured under Se-deficient, Se-adequate, or Se-supranutritional conditions to the Nrf2 activators sulforaphane, cardamonin, or diethyl maleate. Nrf2 protein levels and intracellular localization were determined by immunoblotting, and mRNA levels of Nrf2 target genes and selenoproteins were assessed by qRT-PCR. Exposure to electrophiles resulted in rapid induction of Nrf2 and its enrichment in the nucleus, independent of the cellular Se status. All three electrophilic compounds caused an enhanced expression of Nrf2 target genes, although with differences regarding extent and time course of their induction. Whereas Se status did not significantly affect mRNA levels of the Nrf2 target genes, gene expression of selenoproteins with a low position in the cellular “selenoprotein hierarchy”, such as glutathione peroxidase 1 (GPX1) or selenoprotein W (SELENOW), was elevated under Se-supplemented conditions, as compared to cells held in Se-deficient media. In conclusion, no major effect of Se status on Nrf2 signalling was observed in HepG2 cells.

scholarly journals Antineoplastic Activity of Chrysin against Human Hepatocellular Carcinoma: New Insight on GPC3/SULF2 Axis and lncRNA-AF085935 Expression

2020 ◽  
Vol 21 (20) ◽  
pp. 7642
Author(s):  
Iman O. Sherif ◽  
Laila A. Al-Mutabagani ◽  
Dina Sabry ◽  
Nehal M. Elsherbiny
Keyword(s):  
Hepatocellular Carcinoma ◽  
Antiproliferative Activity ◽  
Hepg2 Cells ◽  
Time Dependent ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Protein Levels ◽  
The Impact ◽  
First Time ◽  
Common Malignancy

The natural flavonoid chrysin possesses antiproliferative activity against various types of cancers, including hepatocellular carcinoma (HCC), which is a common malignancy. However, the exact mechanism of chrysin antiproliferative activity remains unclear. This research was executed to explore the impact of chrysin on glypican-3 (GPC3)/sulfatase-2 (SULF2) axis and lncRNA-AF085935 expression in HCC using HepG2 cells. Cisplatin (20, 50, 100 μg/mL), chrysin (15, 30, and 60 μg/mL) and the combination of 50 μg/mL cisplatin with different concentrations of chrysin were applied for 24/48 h. Cell viability was determined by MTT assay. Protein levels of GPC3 and SULF2 were measured by ELISA at 24/48 h. GPC3 immunoreactivity was detected by immunocytochemistry. Moreover, GPC3 and SULF2 mRNA expressions in addition to lncRNA-AF085935 expression were assessed by qPCR at 48 h. The GPC3 protein, immunostaining and mRNA levels, SULF2 protein and mRNA levels, as well as lncRNA-AF085935 expression, were decreased significantly with cisplatin and chrysin alone when compared with the control untreated HepG2 cells. However, the combination treatment exhibited a better chemopreventive effect in a dose- and time-dependent manner. This study demonstrated, for the first time, the antiproliferative activity of chrysin against HCC through the suppression of the GPC3/SULF2 axis along with the downregulation of lncRNA-AF085935 expression. Synergistic effect of chrysin with cisplatin could potentiate their antiproliferative action in a dose- and time-dependent manner.

scholarly journals The NLRP3 inflammasome mediates liver failure by activating procaspase-1 and pro-IL-1 β and regulating downstream CD40-CD40L signaling

2021 ◽  
Vol 49 (9) ◽  
pp. 030006052110368
Author(s):  
Zenghui Li ◽  
Jianning Jiang
Keyword(s):  
Hepatitis B ◽  
Liver Failure ◽  
Nlrp3 Inflammasome ◽  
Case Control Study ◽  
Case Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Protein Levels ◽  
Control Study ◽  
Prospective Case Control Study

Objectives In this prospective case–control study, we explored the regulatory roles of the NLRP3 inflammasome in hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF). Methods Thirty patients with HBV-ACLF, 30 patients with chronic hepatitis B, and 30 healthy individuals were enrolled. Real-time reverse transcription polymerase chain reaction was used to assess mRNA levels in peripheral blood mononuclear cells and serum protein levels were assessed by enzyme-linked immunosorbent assay. Results Serum levels of alanine aminotransferase, asparagine aminotransferase, total bilirubin, and direct bilirubin in patients with HBV-ACLF were increased. Transcript levels of NLRP3 and ASC and protein levels of interleukin (IL)-1β, IL-18, and sCD40L were elevated in patients with HBV-ACLF. Expression of the NLRP3 inflammasome signaling pathway components procaspase-1 and pro-IL-1β was elevated in patients with HBV-ACLF. Conclusions This prospective case-control study demonstrated that significant activation of the NLRP3 inflammasome occurs in patients with HBV-ACLF. The activated NLRP3 inflammasome mediated liver failure by stimulating procaspase-1 and pro-IL-1 β and regulating downstream CD40-CD40L signaling.

Hydrogen sulfide accumulates LDL receptor precursor via downregulating PCSK9 in HepG2 cells

2020 ◽  
Vol 319 (6) ◽  
pp. C1082-C1096
Author(s):  
Yong Huang ◽  
Ke Ning ◽  
Wen-Wen Li ◽  
Ge Lin ◽  
Cui-Lan Hou ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Transcriptional Activation ◽  
Hepg2 Cells ◽  
Mrna Level ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Mrna Levels ◽  
Sodium Hydrosulfide ◽  
Protein Levels

Endogenous hydrogen sulfide (H2S) affects cholesterol homeostasis and liver X receptor α (LXRα) expression. However, whether low-density lipoprotein (LDL) receptor (LDLR), a key player in cholesterol homeostasis, is regulated by exogenous H2S through LXRα signaling has not been determined. We investigated the effects of sodium hydrosulfide (NaHS, H2S donor) on LDLR expression in the presence or absence of LXR agonists, T0901317 or GW3965 in HepG2 cells. We found that H2S strongly accumulated LDLR precursor in the presence of T0901317. Hence, LDLR transcription and the genes involved in LDLR precursor maturation and degradation were studied. T0901317 increased the LDLR mRNA level, whereas H2S did not affect LDLR transcription. H2S had no significant effect on the expression of LXRα and inducible degrader of LDLR (IDOL). H2S and T0901317 altered mRNA levels of several enzymes for N- and O-glycosylation and endoplasmic reticulum (ER) chaperones assisting LDLR maturation, but did not affect their protein levels. H2S decreased proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels and its mRNA level elevated by T0901317. T0901317 with PCSK9 siRNA also accumulated LDLR precursor as did T0901317 with H2S. High glucose increased PCSK9 protein levels and attenuated LDLR precursor accumulation induced by T0901317 with H2S. Taken together, H2S accumulates LDLR precursor by downregulating PCSK9 expression but not through the LXRα-IDOL pathway, LDLR transcriptional activation, or dysfunction of glycosylation enzymes and ER chaperones. These results also indicate that PCSK9 plays an important role in LDLR maturation in addition to its well-known effect on the degradation of LDLR mature form.

scholarly journals LPS Induces Occludin Dysregulation in Cerebral Microvascular Endothelial Cells via MAPK Signaling and Augmenting MMP-2 Levels

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Lan-hui Qin ◽  
Wen Huang ◽  
Xue-an Mo ◽  
Yan-lan Chen ◽  
Xiang-hong Wu
Keyword(s):  
Endothelial Cells ◽  
Central Nervous System Infection ◽  
Mapk Signaling ◽  
Microvascular Endothelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Doxycycline Hyclate ◽  
Protein Levels ◽  
Cell Vitality ◽  
Microvascular Endothelial

Disrupted blood-brain barrier (BBB) integrity contributes to cerebral edema during central nervous system infection. The current study explored the mechanism of lipopolysaccharide- (LPS-) induced dysregulation of tight junction (TJ) proteins. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to LPS, SB203580 (p38MAPK inhibitor), or SP600125 (JNK inhibitor), and cell vitality was determined by MTT assay. The proteins expressions of p38MAPK, JNK, and TJs (occludin and zonula occludens- (ZO-) 1) were determined by western blot. The mRNA levels of TJ components and MMP-2 were measured with quantitative real-time polymerase chain reaction (qRT-PCR), and MMP-2 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). LPS, SB203580, and SP600125 under respective concentrations of 10, 7.69, or 0.22 µg/mL had no effects on cell vitality. Treatment with LPS decreased mRNA and protein levels of occludin and ZO-1 and enhanced p38MAPK and JNK phosphorylation and MMP-2 expression. These effects were attenuated by pretreatment with SB203580 or SP600125, but not in ZO-1 expression. Both doxycycline hyclate (a total MMP inhibitor) and SB-3CT (a specific MMP-2 inhibitor) partially attenuated the LPS-induced downregulation of occludin. These data suggest that MMP-2 overexpression and p38MAPK/JNK pathways are involved in the LPS-mediated alterations of occludin in hCMEC/D3; however, ZO-1 levels are not influenced by p38MAPK/JNK.

scholarly journals Asian plantain (Plantago asiatica) essential oils suppress 3-hydroxy-3-methyl-glutaryl-co-enzyme A reductase expression in vitro and in vivo and show hypocholesterolaemic properties in mice

2007 ◽  
Vol 99 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Mi Ja Chung ◽  
Kuen Woo Park ◽  
Kyoung Heon Kim ◽  
Cheong-Tae Kim ◽  
Jun Pill Baek ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Ldl Receptor ◽  
Ldl Oxidation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Protein Levels ◽  
Plantago Asiatica ◽  
Coa Reductase

Asian plantain (Plantago asiatica) essential oil (PAEO) contains multiple bioactive compounds, but its potential effects on lipid metabolism have not been examined. PAEO was found to be mostly composed of oxygenated monoterpenes, with linalool as the major component (82·5 %, w/w), measured using GC–MS. Incubation of 0–200 μg PAEO/ml with HepG2 cells for 24 h resulted in no significant toxicity. Incubation with 0·2 mg PAEO/ml altered the expression of LDL receptor (+83 %; P < 0·05) and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase ( − 37 %; P < 0·05), as assessed using RT-PCR. LDL oxidation was markedly inhibited by PAEO treatment due to the prevalence of linalool compounds in PAEO. Oral administration of PAEO for 3 weeks in C57BL/6 mice significantly reduced plasma total cholesterol and TAG concentrations by 29 and 46 %, respectively. The mRNA (+58 %; P < 0·05), but not protein, levels of the LDL receptor were significantly higher, whereas both mRNA and protein levels of HMG-CoA reductase were significantly lower ( − 46 and − 11 %, respectively; P < 0·05) in the liver of PAEO-fed than of control mice. The mRNA levels of CYP7A1 were marginally reduced in HepG2 cells, but not in mouse liver after PAEO treatment. Thus, PAEO may have hypocholesterolaemic effects by altering the expression of HMG-CoA reductase. Reduced TAG and oxidised LDL may provide additional cardiovascular protective benefits.

scholarly journals Interleukin-6 (IL-6) and IL-8 Are Induced in Human Oral Epithelial Cells in Response to Exposure to PeriodontopathicEikenella corrodens

1999 ◽  
Vol 67 (1) ◽  
pp. 384-394 ◽  
Author(s):  
Hiromichi Yumoto ◽  
Hideaki Nakae ◽  
Keiko Fujinaka ◽  
Shigeyuki Ebisu ◽  
Takashi Matsuo
Keyword(s):  
Epithelial Cells ◽  
Bacterial Colonization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Kb Cells ◽  
Protein Levels ◽  
Periodontal Tissues ◽  
Oral Epithelial Cells ◽  
Oral Epithelial ◽  
Stimulation Of

ABSTRACT Periodontitis is the inflammatory response in periodontal tissues elicited by bacterial colonization in periodontal pockets. In this response, pocket epithelial cells are the first cells to come into contact with bacteria. To elucidate this mechanism, we determined the adherence of the periodontopathic bacterium Eikenella corrodens 1073, which has a GalNAc-sensitive lectin-like adhesin (EcLS), to a human oral epithelial carcinoma cell line (KB) and the induction of proinflammatory cytokine production in the cells following exposure to this bacterium in vitro. In the adherence assay, EcLS played a role as the adhesin of this bacterium in adherence to KB cells. In a reverse transcriptase PCR, significant interleukin-8 (IL-8) and IL-6 mRNA levels were induced in response to exposure to this bacterium. In an enzyme-linked immunosorbent assay after an 8-h bacterial exposure, the IL-8 and IL-6 protein levels were 13.5- and 8.3-fold higher than those in the nonexposed controls, respectively. These protein responses were time dependent. Interestingly, whenE. corrodens was separated from KB cells by cell culture inserts, a slight stimulation of the IL-6 and IL-8 mRNA and secreted protein levels was seen. These results imply that the direct contact ofE. corrodens 1073 with oral epithelial cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion. We suggest that E. corrodens induces the epithelial cells to secrete proinflammatory cytokines which serve as an early signaling system to host immune and inflammatory cells in underlying connective tissues.

Effects of calcium hydroxide and N-acetylcysteine on MMP-2, MMP-9, TIMP-1 and TIMP-2 in LPS-stimulated macrophage cell lines

2017 ◽  
Vol 43 (6) ◽  
pp. 571-577
Author(s):  
Eda Ezgi Aslantaş ◽  
Yasemin Aksoy ◽  
Yeliz Zülfiye Akkaya Ulum ◽  
Deniz Ceyhan ◽  
Banu Peynircioglu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Calcium Hydroxide ◽  
Human Monocyte ◽  
Matrix Metalloproteinase 2 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Macrophage Cell ◽  
Protein Levels ◽  
Bonferroni Test ◽  
Polymerase Chain

Abstract Aim This study was evaluated the effects of N-acetylcysteine (NAC) and calcium hydroxide (Ca(OH)2) on the expression levels of matrix metalloproteinase -2, -9 (MMP-2, -9) and tissue inhibitor metalloproteinase -1, -2 (TIMP-1, -2) in lipopolysaccharide (LPS)-stimulated human macrophages. Methods Human monocyte precursor cells (THP-1) were differentiated into macrophage-adherent cells and were stimulated with LPS for 24 h. Then individually incubated with NAC or Ca(OH)2 for 24, 48 and 72 h. Following incubation, protein expression and mRNA levels of MMP-2, -9 and TIMP-1, -2 were evaluated using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Data were statistically analysed using two-way ANOVA, to followed by Bonferroni test at α=0.05. Results NAC significantly decreased mRNA expression and protein levels of MMP-9, while Ca(OH)2 decreased mRNA expression alone at 24 h. NAC and Ca(OH)2 decreased mRNA expression of MMP-2 at 24 h, while NAC increased this expression at 48 h. Although NAC and Ca(OH)2 decreased the mRNA expression of TIMP-1, -2 at 24 h, only NAC increased mRNA expression of TIMP-1 at 48 h. Conclusion At the early stages of inflammation, NAC and Ca(OH)2 have anti-inflammatory effects on macrophages.

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