scholarly journals Regulation of Cytochrome P450 2a5 by Artemisia capillaris and 6,7-Dimethylesculetin in Mouse Hepatocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Sangsoo Daniel Kim ◽  
Larry Morgan ◽  
Elyse Hargreaves ◽  
Xiaoying Zhang ◽  
Zhihui Jiang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Nuclear Translocation ◽  
Constitutive Androstane Receptor ◽  
Elevated Serum ◽  
Regulatory Control ◽  
Herbal Remedies ◽  
Luciferase Reporter ◽  
Artemisia Capillaris ◽  
Primary Mouse ◽  
Mouse Hepatocytes

Jaundice is a potentially fatal condition resulting from elevated serum bilirubin levels. For centuries, herbal remedies containing Artemisia capillaris Thunb. including the compound 6,7-dimethylesculetin (DE) have been used in Asia to prevent and treat jaundice in neonates. DE activates an important regulator of bilirubin metabolism, the constitutive androstane receptor (CAR), and increases bilirubin clearance. In addition, murine cytochrome P450 2a5 (Cyp2a5) is known to be involved in the oxidative metabolism of bilirubin. Moreover, treatment of mice with phenobarbital, a known inducer of both CAR and Cyp2a5, increases expression of Cyp2a5 suggesting a potential relationship between CAR and Cyp2a5 expression. The aim of this study is to investigate the influence of Artemisia capillaris and DE on the expression and regulatory control of Cyp2a5 and the potential involvement of CAR. Treatment of mouse hepatocytes in primary culture with DE (50 μM) significant increased Cyp2a5 mRNA and protein levels. In mice, Artemisia capillaris and DE treatment also increased levels of hepatic Cyp2a5 protein. Luciferase reporter assays showed that CAR increases Cyp2a5 gene transcription through a CAR response element in the Cyp2a5 gene promoter. Moreover, DE caused nuclear translocation of CAR in primary mouse hepatocytes and increased Cyp2a5 transcription in the presence of CAR. These results identify a potential CAR-mediated mechanism by which DE regulates Cyp2a5 gene expression and suggests that DE may enhance bilirubin clearance by increasing Cyp2a5 levels. Understanding this process could provide an opportunity for the development of novel therapies for neonatal and other forms of jaundice.

scholarly journals Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Anayelly López-Islas ◽  
Victoria Chagoya-Hazas ◽  
Benjamin Pérez-Aguilar ◽  
Mayrel Palestino-Domínguez ◽  
Verónica Souza ◽  
...  
Keyword(s):  
Er Stress ◽  
Toxic Effect ◽  
Nuclear Translocation ◽  
Cholesterol Content ◽  
Primary Hepatocytes ◽  
Transmission Electron ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Stress Oxidative

Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

scholarly journals Role of miR-383 and miR-146b in different propensities to obesity in male mice

2017 ◽  
Vol 234 (2) ◽  
pp. 201-216 ◽  
Author(s):  
Shu-Fang Xia ◽  
Xiao-Mei Duan ◽  
Xiang-Rong Cheng ◽  
Li-Mei Chen ◽  
Yan-Jun Kang ◽  
...  
Keyword(s):  
Hepatic Steatosis ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Stem Loop ◽  
Next Generation Sequencing Technology ◽  
Diet Induced Obesity ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Cellular Lipid Accumulation

The study was designed to investigate the possible mechanisms of hepatic microRNAs (miRs) in regulating local thyroid hormone (TH) action and ultimately different propensities to high-fat diet (HFD)-induced obesity. When obesity-prone (OP) and obesity-resistant (OR) mice were fed HFD for 7 weeks, OP mice showed apparent hepatic steatosis, with significantly higher body weight and lower hepatic TH receptor b (TRb) expression and type 1 deiodinase (DIO1) activity than OR mice. Next-generation sequencing technology revealed that 13 miRs in liver were dysregulated between the two phenotypes, of which 8 miRs were predicted to target on Dio1 or TRb. When mice were fed for 17 weeks, OR mice had mild hepatic steatosis and increased Dio1 and TRb expression than OP mice, with downregulation of T3 target genes (including Srebp1c, Acc1, Scd1 and Fasn) and upregulation of Cpt1α, Atp5c1, Cox7c and Cyp7a1. A stem-loop qRT-PCR analysis confirmed that the levels of miR-383, miR-34a and miR-146b were inversely correlated with those of DIO1 or TRb. Down-regulated expression of miR-383 or miR-146b by miR-383 inhibitor (anti-miR-383) or miR-146b inhibitor (anti-miR-146b) in free fatty acid-treated primary mouse hepatocytes led to increased DIO1 and TRb expressions, respectively, and subsequently decreased cellular lipid accumulation, while miR-34a inhibitor (anti-miR-34a) transfection had on effects on TRb expression. Luciferase reporter assay illustrated that miR-146b could directly target TRb 3′untranslated region (3′UTR). These findings suggested that miR-383 and miR-146b might play critical roles in different propensities to diet-induced obesity via targeting on Dio1 and TRb, respectively.

scholarly journals Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2532
Author(s):  
Josef Skoda ◽  
Jan Dusek ◽  
Martin Drastik ◽  
Alzbeta Stefela ◽  
Klara Dohnalova ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Constitutive Androstane Receptor ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Humanized Mice ◽  
Luciferase Reporter ◽  
Heparg Cells ◽  
Reporter Assays

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

Proteome analysis of 3,5-T2-treated primary mouse hepatocytes

2018 ◽  
Author(s):  
Janine Golchert ◽  
Julika Lietzow ◽  
Uwe Volker ◽  
Georg Homuth ◽  
Josef Kohrle
Keyword(s):  
Proteome Analysis ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

2020 ◽  
Vol 25 (45) ◽  
pp. 4806-4812 ◽  
Author(s):  
Zhibo Sun ◽  
Fei Wu ◽  
Yue Yang ◽  
Feng Liu ◽  
Fengbo Mo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Translocation ◽  
Bone Diseases ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Alizarin Red ◽  
Over Expression ◽  
Negative Effect ◽  
Proliferation Ability

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

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