scholarly journals Liraglutide Inhibits Hepatitis C Virus Replication Through an AMP Activated Protein Kinase Dependent Mechanism

2019 ◽  
Vol 20 (18) ◽  
pp. 4569 ◽  
Author(s):  
Mei-Yueh Lee ◽  
Wei-Chun Chen ◽  
Wei-Hao Hsu ◽  
Szu-Chia Chen ◽  
Jin-Ching Lee
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Adenosine Monophosphate ◽  
Hcv Rna ◽  
Dependent Manner

Insulin resistance and diabetes are both associated with chronic hepatitis C virus (HCV) infection, and the glucagon-like peptide-1(GLP-1) receptor agonist, liraglutide, is a common therapy for diabetes. Our aim was to investigate whether liraglutide treatment can inhibit HCV replication. A cell culture-produced HCV infectious system was generated by transfection of in vitro-transcribed genomic JFH-1 ribonucleic acid (RNA) into Huh-7.5 cells. Total RNA samples were extracted to determine the efficiency of HCV replication. The Ava5 cells were treated with liraglutide and cell viability was calculated. A Western blot analysis of the protein expression was performed. The immunoreactive blot signals were also detected. Liraglutide activated GLP-1 receptors in the HCV infectious system, and inhibited subgenomic HCV RNA replication in the HuH-7.5 cells. The Western blot analysis revealed both HCV protein and replicon RNA were reduced after treatment with liraglutide in a dose-dependent manner. Liraglutide decreased the cell viability of HCV RNA at an optimum concentration of 120 μg/mL, activated the 5′ adenosine monophosphate-activated protein kinase (AMPK) and the phosphorylated- transducer of regulated cyclic adenosine monophosphate (CAMP) response element-binding protein 2 (TORC2), thereby decreasing the cell viability of phosphoenolpyruvate carboxykinase (PEPCK) and G6pase RNA Therefore, we conclude that liraglutide can inhibit HCV replication via an AMPK/TORC2-dependent pathway.

scholarly journals Glycyrrhizin inhibits LPS-induced inflammatory responses in goat ruminal epithelial cells in vitro

2020 ◽  
Author(s):  
Xuanxuan Pu ◽  
Pattygouri Mullahred ◽  
Junfeng Liu ◽  
Xuefeng Guo ◽  
Jian Gao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Cell Viability ◽  
Mrna Expression ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Staining ◽  
Dependent Manner ◽  
Relative Mrna Expression

AbstractA long-term of high concentration feeding in ruminants can bring huge economic profits, but it also impose ruminants into great threat of suffering subacute ruminal acidosis (SARA). SARA is a kind of disease which attenuate the health, feed intake and production of ruminants, and when ruminants suffer SARA, the concentration of lipopolysaccharide (LPS) increase largely. Glycyrrhizin is reported to have anti-inflammation effects, and the study was conducted to investigate effects of glycyrrhizin on LPS-induced goat ruminal epithelial cells (GRECs) to provide evidence for using glycyrrhizin as a treatment for SARA. Effects of LPS, and glycyrrhizin on cell viability of GRECs were investigated, respectively. Then GRECs were stimulated with LPS (50 mg/L) for 2 h, and glycyrrhizin were added at the concentration of 0, 50, 75, 100 and 125 mg/L for 24 h to investigate the expression of inflammatory cytokines (by Elisa kits), the mRNA expression of NF-κB and inflammatory cytokines (by qRT-PCR), the distribution of Zo-1 and Occludin (by immunofluorescence staining), the expression of Occludin (by Western blot analysis), and the morphology of GRECs. The results showed that: (1) Glycyrrhizin at the concentration of 50, 75, 100, and 125 mg/L had no cytotoxic effects on GRECs, and LPS at the concentration of 50 mg/L significantly decreased the cell viability of GRECs. (2) Glycyrrhizin attenuated the expression and relative mRNA expression of TNF-α, IL-1β, IL-6, IL-8 and IL-12 by a dose-dependent manner, and significantly attenuated the relative mRNA expression of NF-κB. (3) Immunofluorescence staining and Western blot analysis showed that the quantity of Zo-1 and Occludin, and the expression of Occludin all increased with the treatment of glycyrrhizin. (4) Glycyrrhizin attenuated LPS-induced autophagy and protected the structural integrity of GRECs. In conclusion, glycyrrhizin significantly inhibited the inflammatory response in LPS-stimulated GRECs, and it may be used as a potential agent for the treatment of SARA.

scholarly journals Immunoproteasome induction is suppressed in hepatitis C virus-infected cells in a protein kinase R-dependent manner

2016 ◽  
Vol 48 (11) ◽  
pp. e270-e270 ◽  
Author(s):  
In Soo Oh ◽  
Kathrin Textoris-Taube ◽  
Pil Soo Sung ◽  
Wonseok Kang ◽  
Xenia Gorny ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Protein Kinase R ◽  
Infected Cells

Abstract 2671: Thymosin Beta 4 Induced Oligodendrogenesis Is Associated With Upregulation Of Serum Response Factor

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Daniel C Morris ◽  
Benjamin Buller ◽  
Manoranjan Santra ◽  
Michael Chopp ◽  
Zheng Gang Zhang
Keyword(s):  
Western Blot ◽  
Progenitor Cells ◽  
Rat Model ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Embolic Stroke ◽  
Early Gene ◽  
Dependent Manner ◽  
Thymosin Beta 4 ◽  
Serum Response

Background: Thymosin beta 4 (Tβ4) is a G-actin sequestering peptide that improves neurological functional outcome when administered 24 hours after onset of stroke to a rat model of embolic stroke. Tβ4 increases the number of oligodendrocyte progenitor cells (OPCs) as well as mature oligodendrocytes (OLs). Mechanisms of Tβ4 induced oligodendrogenesis (OLG) remain unclear. Serum response growth factor (SRF) is a transcriptional factor which binds with ternary complex co-factors to primarily convey an immediate early gene response to influence and orchestrate neuronal migration and differentiation. Hypothesis: We tested the hypothesis that Tβ4 upregulates SRF with subsequent increase in the markers of OL differentiation. Results: We employed a mouse OPC line (N20.1) to investigate the mechanisms of Tβ4-induced OLG. The cells were plated at a density of 100,000 cells/ml and grown in the presence of 0, 12.5, 25 and 50 ng/ml of Tβ4 (RegeneRx Biopharmaceuticals, Inc.) for 14 days (n=3). Western blot analysis revealed that SRF was dose-dependently upregulated by a factor of 4. Quantitative real time PCR and Western blot analysis showed that Tβ4 treatment induced myelin basic protein (MBP) and 2’, 3’-cyclic nucleotide, 3’-phosphodiesterase (CNPase) expression in a dose-dependent manner by ∼2 fold, indicating the stimulation of OLG. In order to independently demonstrate that SRF promotes the differentiation of progenitor cells into mature oligodendrocytes, SRF was over expressed in the N20.1 cells using a plasmid encoding the SRF gene. After six days SRF over expressed N20.1 cells (n=3) demonstrated an increase of expression of MBP (26 ± 3%) and CNPase (23 ± 3%) when compared to cells transfected with an empty expression plasmid (n=3, MBP, 14 ± 3% and CNPase, 10 ± 4%, p<0.05). Conclusions: In this mouse model of OPCs, SRF was upregulated by Tβ4 and may be involved in Tβ4 induced OLG. Further in vivo investigation of SRF is warranted in our rat model of embolic stroke.

scholarly journals NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner

2006 ◽  
Vol 87 (6) ◽  
pp. 1703-1713 ◽  
Author(s):  
Lin Deng ◽  
Motoko Nagano-Fujii ◽  
Motofumi Tanaka ◽  
Yuki Nomura-Takigawa ◽  
Masanori Ikeda ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Subcellular Localization ◽  
Serine Protease ◽  
Protease Activity ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Diffuse Type ◽  
Serine Protease Activity ◽  
Tumour Suppressor P53

The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.

scholarly journals MicroRNA-145 attenuates IL-6-induced enhancements of sensitivity to UVB irradiation by suppressing MyD88 in HaCaT cells

2018 ◽  
Vol 32 ◽  
pp. 205873841879594 ◽  
Author(s):  
Hui Dong ◽  
Wei Jiang ◽  
Hongquan Chen ◽  
Shui Jiang ◽  
Yunshu Zang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Lupus Erythematosus ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Ultraviolet B ◽  
Hacat Cells ◽  
Related Factors ◽  
Uvb Irradiation

MicroRNAs (miRNAs/miRs) play vital roles in various immune diseases including systemic lupus erythematosus (SLE). The current study aimed to assess the role of miR-145 in interleukin-6 (IL-6)-treated HaCaT cells under ultraviolet B (UVB) irradiation and further explore the potential regulatory mechanism. HaCaT cells were pretreated with IL-6 and then exposed to UVB to assess the effect of IL-6 on sensitivity of HaCaT cells to UVB irradiation. The levels of miR-145 and MyD88 were altered by transfection and the transfected efficiency was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR)/western blot analysis. Cell viability, percentage of apoptotic cells and expression levels of apoptosis-related factors were measured by trypan blue assay, flow cytometry assay, and western blot analysis, respectively. In addition, the levels of c-Jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) signaling pathway-related factors were assessed by western blot analysis. IL-6 treatments significantly aggravated the reduction of cell viability and promotion of cell apoptosis caused by UVB irradiation in HaCaT cells. Interestingly, miR-145 level was augmented by UVB exposure and miR-145 mimic alleviated IL-6-induced increase of sensitivity to UVB irradiation in HaCaT cells, as dramatically increased cell viability and reduced cell apoptosis. Opposite effects were observed in miR-145 inhibitor-transfected cells. Meanwhile, MyD88 was negatively regulated by miR-145 and MyD88 mediated the regulatory effect of miR-145 on IL-6- and UVB-treated cells. In addition, miR-145 mimic inhibited the JNK and NF-κB pathways by down-regulating MyD88. In conclusion, the present study demonstrated that miR-145 alleviated IL-6-induced increase of sensitivity to UVB irradiation by down-regulating MyD88 in HaCaT cells.

scholarly journals MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Isaac Ruiz ◽  
Quentin Nevers ◽  
Eva Hernández ◽  
Nazim Ahnou ◽  
Rozenn Brillet ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatoma Cell ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Cysteinyl Leukotriene ◽  
Leukotriene Receptor ◽  
Cysteinyl Leukotriene Receptor ◽  
Dose Dependent ◽  
Rna Levels

ABSTRACT The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10. MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

scholarly journals The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein

2004 ◽  
Vol 381 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Hongbing LI ◽  
Juan SÁNCHEZ-TORRES ◽  
Alan del CARPIO ◽  
Valentina SALAS ◽  
Antonio VILLALOBO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Tyrosine Kinases ◽  
Western Blot Analysis ◽  
Binding Protein ◽  
Signalling Pathways ◽  
Breast Adenocarcinoma ◽  
Dependent Manner ◽  
Her2 Receptor

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM–agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

scholarly journals Conventional Protein Kinase C Inhibition Prevents Alpha Interferon-Mediated Hepatitis C Virus Replicon Clearance by Impairing STAT Activation

2004 ◽  
Vol 78 (23) ◽  
pp. 12809-12816 ◽  
Author(s):  
Gian Maria Fimia ◽  
Cristina Evangelisti ◽  
Tonino Alonzi ◽  
Marta Romani ◽  
Federica Fratini ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Rna Degradation ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Kinase C ◽  
Limited Effectiveness

ABSTRACT Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. The only treatment available for HCV infections, alpha interferon (IFN-α), is effective in a limited percentage of patients. The mechanisms by which IFN-α interferes with the HCV life cycle and the reasons for limited effectiveness of IFN-α therapy have not yet been fully elucidated. Using a cell-based HCV replication system and specific kinase inhibitors, we examined the role played by various signaling pathways in the IFN-α-mediated HCV clearance. We reported that conventional protein kinase C (cPKC) activity is important for the effectiveness of IFN-α treatment. In cells treated with a cPKC-specific inhibitor, IFN-α failed to induce an efficient HCV RNA degradation. The lack of cPKC activity leads to a broad reduction of IFN-α-stimulated gene expression due to a significant impairment of STAT1 and STAT3 tyrosine phosphorylation. Thus, modulation of cPKC function by either host or viral factors could influence the positive outcome of IFN-α-mediated antiviral therapies.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

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