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American Journal of Physiology.... Jul 2010Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids...
Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.
Topics: 5' Flanking Region; Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Butylamines; Chromatin Immunoprecipitation; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Genes, Reporter; Hep G2 Cells; Hepatocytes; Humans; Methylcholanthrene; Mice; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Mutation; NF-E2-Related Factor 2; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Pyrazines; RNA Interference; RNA, Messenger; Receptors, Aryl Hydrocarbon; Response Elements; Thiones; Thiophenes; Time Factors; Transfection
PubMed: 20395535
DOI: 10.1152/ajpgi.00522.2010 -
Endocrinology Feb 2018We investigated the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in renin-angiotensin system (RAS) gene expression in renal proximal tubule cells (RPTCs)...
Nrf2 Deficiency Upregulates Intrarenal Angiotensin-Converting Enzyme-2 and Angiotensin 1-7 Receptor Expression and Attenuates Hypertension and Nephropathy in Diabetic Mice.
We investigated the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in renin-angiotensin system (RAS) gene expression in renal proximal tubule cells (RPTCs) and in the development of systemic hypertension and kidney injury in diabetic Akita mice. We used adult male Akita Nrf2 knockout mice and Akita mice treated with trigonelline (an Nrf2 inhibitor) or oltipraz (an Nrf2 activator). We also examined rat immortalized RPTCs (IRPTCs) stably transfected with control plasmids or plasmids containing rat angiotensinogen (Agt), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (Ace2), or angiotensin 1-7 (Ang 1-7) receptor (MasR) gene promoters. Genetic deletion of Nrf2 or pharmacological inhibition of Nrf2 in Akita mice attenuated hypertension, renal injury, tubulointerstitial fibrosis, and the urinary albumin/creatinine ratio. Furthermore, loss of Nrf2 upregulated RPTC Ace2 and MasR expression, increased urinary Ang 1-7 levels, and downregulated expression of Agt, ACE, and profibrotic genes in Akita mice. In cultured IRPTCs, Nrf2 small interfering RNA transfection or trigonelline treatment prevented high glucose stimulation of Nrf2 nuclear translocation, Agt, and ACE transcription with augmentation of Ace2 and MasR transcription, which was reversed by oltipraz. These data identify a mechanism, Nrf2-mediated stimulation of intrarenal RAS gene expression, by which chronic hyperglycemia induces hypertension and renal injury in diabetes.
Topics: Angiotensin I; Angiotensin-Converting Enzyme 2; Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gene Expression Regulation, Enzymologic; Hypertension; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-E2-Related Factor 2; Peptide Fragments; Peptidyl-Dipeptidase A; Rats; Receptor, Angiotensin, Type 2; Renin-Angiotensin System; Up-Regulation
PubMed: 29211853
DOI: 10.1210/en.2017-00752 -
Bulletin of Emergency and Trauma Oct 2014To determine the effects of topical administration of 20% oltipraz solution on histomorphometrical and stereological aspects of skin tissue in full thickness skin wounds...
OBJECTIVE
To determine the effects of topical administration of 20% oltipraz solution on histomorphometrical and stereological aspects of skin tissue in full thickness skin wounds in laboratory rats.
METHODS
Thirty-six male Wistar portion rats (220±20 g) were randomly divided into three groups (n=12). On the first day of experimentation, a 1-cm2 circular wound was made on the posterior surface of neck in all rats by removing a full thickness skin piece immediately after induction of anesthesia with ether inhalation. One group was treated with vehicle solution (DMSO alone). The second group was treated daily with 20% oltipraz solution, and the third group, the control group, received no treatment. The wound closure rate was estimated our previously described method. The volume density of collagen bundles, vessels, and hair follicles, the vessels' length density, mean diameter of vessels and also fibroblast population were estimated by using stereological methods.
RESULTS
The oltipraz group indicated a significantly higher improvement (6.26% of the wound surface per day) than control and the vehicle treated groups (p=0.032); furthermore, there was inconsiderable difference between the rate of wound closure in the group treated with vehicle (4.93% per day) and the control group (4.43% per day).
CONCLUSION
Oltipraz has positive influence on fibroblast proliferation and re-pithelization. A noticeable observation in our study was absence of scar formation in wounds which were treated by oltipraz and can be mentioned as an advantage of this drug.
PubMed: 27162890
DOI: No ID Found -
BMC Ophthalmology Dec 2018Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic...
BACKGROUND
Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic UV exposure is considered as a pathogenic factor of this disease. Proteasome is an intracellular multi-subunit protease complex that degrades intracellular proteins. Among proteasome subunits the β5 (PSMB5), bearing chymotrypsin-like activity. It is considered as the main proteasome subunit and its expression is mediated by Nrf2-ARE pathway in many cell types. This study investigates the expression of PSMB5 in pterygium and the effect of UVB irradiation on its expression and activity in pterygium fibroblasts.
METHODS
Normal conjunctival and pterygium specimens were obtained from the bulbar conjunctiva of patients undergoing cataract surgery and from patients with pterygium undergoing surgical removal of primary tissue, respectively. Fibroblasts were isolated upon treatment of specimens with clostridium collagenase. The expression of PSMB5 and Nrf2 in tissues and cells was ascertained by RT-PCR analysis and western blotting. Cell survival was measured by the MTT method and the proteasome chymotrypsin-like activity was determined by fluorometry.
RESULTS
RT-PCR analysis showed that the expression of PSMB5 was significantly lower in pterygium than in normal conjunctiva. The expression of PSMB5 was mediated by the Nrf2/ARE pathway as indicated by using the Nrf2 activator Oltipraz. The expression of PSMB5 and Nrf2 by pterygium fibroblasts was suppressed in a dose dependent manner following UVB radiation of 0-50 mJ/cm doses. The expression of PSMB5, but not of Nrf2, remained at almost the control levels, when UVB exposure was performed after pre-incubation of cells with the src kinases inhibitor PP2. UVB irradiation had very low deleterious effect on fibroblasts survival, while it did not affect the proteasome chymotrypsin-like activity.
CONCLUSION
In pterygium fibroblasts, UVB exposure leads to down-regulation of Nrf2/ARE-mediated PSMB5 gene expression, in which src kinases may be implicated. This effect may be partially responsible for the lower expression of PSMB5 detected in pterygium as compared to normal conjunctiva.
Topics: Aged; Aged, 80 and over; Cell Survival; Cells, Cultured; Conjunctiva; Down-Regulation; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; Middle Aged; NF-E2-Related Factor 2; Proteasome Endopeptidase Complex; Pterygium; Reverse Transcriptase Polymerase Chain Reaction; Ultraviolet Rays
PubMed: 30563490
DOI: 10.1186/s12886-018-0987-8 -
Aging Mar 2021Human Mesenchymal stem cells (hMSCs) are multi-potential cells which are widely used in cell therapy. However, the frequently emerged senescence and decrease of...
Human Mesenchymal stem cells (hMSCs) are multi-potential cells which are widely used in cell therapy. However, the frequently emerged senescence and decrease of differentiation capabilities limited the broad applications of MSC. Several strategies such as small molecules treatment have been widely studied and used to improve the stem characteristics bypassing the senescence but the exact mechanisms for them to reduce senescence have not been fully studied. In this study, hMSCs were treated by rapamycin, oltipraz, metformin, and vitamin C for the indicated time and these cells were subjected to senescence evaluation and trilineage differentiation. Furthermore, transcriptomics and lipidomics datasets of hMSCs after drug treatment were analyzed to interpret biological pathways responsible for their anti-senescence effects. Although four drugs exhibited significant activities in promoting MSC osteogenic differentiation, metformin is the optimal drug to promote trilineage differentiation. GO terms illustrated that the anti-aging effects of drugs were mainly associated with cellular senescence, mitotic and meiosis process. Biosynthesis of phosphatidylcholines (PC) and phosphatidylethanolamine (PE) were inhibited whereas production of phosphatidylinositols (PIs) and saturated fatty acids (SFA)/ mono-unsaturated fatty acids (MUFA) conversion was activated. Medium free fatty acids (FFA) was increased in hMSCs with different anti-aging phenotypes. Therefore, we established a comprehensive method in assessing drug intervention based on the results of transcriptomics and lipidomics. The method can be used to study different biological phenotypes upon drug intervention in MSC which will extend the clinical application of hMSCs.
Topics: Ascorbic Acid; Cell Differentiation; Cellular Senescence; Humans; Hypoglycemic Agents; Lipidomics; Mesenchymal Stem Cells; Metformin; Pyrazines; Reverse Transcriptase Inhibitors; Sirolimus; Thiones; Thiophenes; Transcriptome
PubMed: 33795523
DOI: 10.18632/aging.202759 -
Cancer Letters Dec 2012Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant...
Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant formulation ("coated" implants) using curcumin, individual curcuminoids, withaferin A and oltipraz. This method involves the coating of blank polycaprolactone implants with 20-30 layers of 10-20% polycaprolactone solution in dichloromethane containing 0.5-2% of the test agent. The in vitro release showed that while oltipraz was released with almost zero-order kinetics over 8 weeks, curcumin, individual curcuminoids and withaferin A were released with some initial burst. The in vivo release was determined by grafting implants subcutaneously in A/J mice. When delivered by coated implants, oltipraz significantly diminished lung DNA adducts in mice treated with dibenzo[a,l]pyrene compared with sham treatment (28 ± 7 versus 54 ± 17 adducts/10(9) nucleotides). Withaferin A also diminished DNA adducts, but it was insignificant. Curcumin and individual curcuminoids were ineffective. Analysis of lung, liver and brain by UPLC-fluorescence showed the presence of the three test curcuminoids indicating effectiveness of the implant delivery system. Further, based on its known antitumor activity in vivo, withaferin A given via the implants significantly inhibited human lung cancer A549 xenograft in athymic nude mice, while it was ineffective when the same total dose was administered i.p. and required over 2-fold higher dose to elicit effectiveness. Together, our data suggest that coated polymeric implants can accommodate heat-labile compounds, can furnish sustained release for long duration, and elicit DNA damage-inhibiting and anti-tumor activities.
Topics: Animals; Anticarcinogenic Agents; Biological Availability; Coated Materials, Biocompatible; Curcumin; DNA Adducts; Female; Humans; Infusion Pumps, Implantable; Mice; Mice, Nude; Polyesters; Polymers; Pyrazines; Thiones; Thiophenes; Tissue Distribution; Withanolides; Xenograft Model Antitumor Assays
PubMed: 22820161
DOI: 10.1016/j.canlet.2012.07.017 -
Carcinogenesis Mar 20093H-1,2-dithiole-3-thione (D3T) and its analogues 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (OLT) and 5-tert-butyl-3H-1,2-dithiole-3-thione (TBD) are chemopreventive...
3H-1,2-dithiole-3-thione (D3T) and its analogues 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (OLT) and 5-tert-butyl-3H-1,2-dithiole-3-thione (TBD) are chemopreventive agents that block or diminish early stages of carcinogenesis by inducing activities of detoxication enzymes. While OLT has been used in clinical trials, TBD has been shown to be more efficacious and possibly less toxic than OLT in animals. Here, we utilize a robust and high-resolution chemical genomics procedure to examine the pharmacological structure-activity relationships of these compounds in livers of male rats by microarray analyses. We identified 226 differentially expressed genes that were common to all treatments. Functional analysis identified the relation of these genes to glutathione metabolism and the nuclear factor, erythroid derived 2-related factor 2 pathway (Nrf2) that is known to regulate many of the protective actions of dithiolethiones. OLT and TBD were shown to have similar efficacies and both were weaker than D3T. In addition, we identified 40 genes whose responses were common to OLT and TBD, yet distinct from D3T. As inhibition of cytochrome P450 (CYP) has been associated with the effects of OLT on CYP expression, we determined the half maximal inhibitory concentration (IC(50)) values for inhibition of CYP1A2. The rank order of inhibitor potency was OLT >> TBD >> D3T, with IC(50) values estimated as 0.2, 12.8 and >100 microM, respectively. Functional analysis revealed that OLT and TBD, in addition to their effects on CYP, modulate liver lipid metabolism, especially fatty acids. Together, these findings provide new insight into the actions of clinically relevant and lead dithiolethione analogues.
Topics: Animals; Male; Rats; Anticarcinogenic Agents; Cytochrome P-450 CYP1A2; Gene Expression Profiling; Genomics; Glutathione; Heterocyclic Compounds, 1-Ring; Liver; Multigene Family; Oligonucleotide Array Sequence Analysis; Pyrazines; Rats, Inbred F344; Structure-Activity Relationship; Thiones; Thiophenes; NF-E2-Related Factor 2
PubMed: 19126641
DOI: 10.1093/carcin/bgn292 -
PloS One 2020Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their... (Comparative Study)
Comparative Study
Comparative effectiveness of 4 natural and chemical activators of Nrf2 on inflammation, oxidative stress, macrophage polarization, and bactericidal activity in an in vitro macrophage infection model.
Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1β, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.
Topics: Anti-Inflammatory Agents; Antioxidants; Dimethyl Fumarate; Escherichia coli; Escherichia coli Infections; Flavanones; Humans; Inflammation; Isothiocyanates; Leukocytes, Mononuclear; Macrophage Activation; Macrophages; NF-E2-Related Factor 2; Oxidative Stress; Pyrazines; Staphylococcal Infections; Staphylococcus aureus; Sulfoxides; THP-1 Cells; Thiones; Thiophenes
PubMed: 32511271
DOI: 10.1371/journal.pone.0234484 -
The Journal of Biological Chemistry Jul 2002Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both...
Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S-transferase expression and activities in rat tissues. Its effects, however, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 microm oltipraz treatment increased CYP1A1 mRNA ( approximately 30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5'-flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic responsive element (XRE). Furthermore we demonstrated that resveratrol, an antagonist of the aryl hydrocarbon (Ah) receptor, inhibited the induction of both CYP1A1 promoter activity and mRNA by oltipraz, supporting the involvement of the Ah receptor in this induction. In an attempt to further characterize the mechanism of CYP1A1 induction, we showed a rapid increase in intracellular calcium concentration upon treatment of Caco-2 cells with oltipraz. Moreover, the effect of this compound on CYP1A1 was strongly abolished in the presence of BAPTA-AM, a well known chelator of intracellular calcium, and 2-aminoethyl diphenylborate, an inhibitor of store-operated calcium channels. These results bring the first demonstration that oltipraz activates transcription of the CYP1A1 gene through the Ah receptor-XRE pathway in Caco-2 cells and that CYP1A1 induction relies upon an increase of intracellular calcium concentration.
Topics: Animals; Anticarcinogenic Agents; Base Sequence; Caco-2 Cells; Colonic Neoplasms; Cytochrome P-450 CYP1A1; DNA Primers; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Male; Molecular Sequence Data; Promoter Regions, Genetic; Pyrazines; RNA, Messenger; Rats; Rats, Wistar; Receptors, Aryl Hydrocarbon; Thiones; Thiophenes; Transcription, Genetic; Tumor Cells, Cultured
PubMed: 11959854
DOI: 10.1074/jbc.M111319200 -
Heliyon Nov 2023Our previous study has confirmed that miR338-3p/TRAP-1 axis was involved in regulation of hyperactivation in human synovial fibroblasts (HFLS) of patients with...
Our previous study has confirmed that miR338-3p/TRAP-1 axis was involved in regulation of hyperactivation in human synovial fibroblasts (HFLS) of patients with osteoarthritis (OA). Here, we aim to further investigate the underlying causes of the abnormal activation miR338-3p/TRAP-1 at the molecular level. Our results showed that the decrease of NF-E2-related factor 2(Nrf2) was the direct cause of downregulation of miR338-3p and upregulation of TRAP-1 protein expression in HFLS of OA patients. Furthermore, we also found that the phosphorylation and nuclear entry of Nrf2 protein were significantly reduced in HFLS of OA patients than that of normal individuals, and both of them were positively correlated with miR338-3p levels. Bioinformatics analysis, luciferase assay, and CHIP experiment together indicated that Nrf2 could positively regulate the transcription of miR338-3p by binding to the Transcription Factor Binding Sites (TFBS) on its promoter. It was confirmed by in vitro assays that oltipraz (agonists of Nrf2) treatment effectively inhibited the hyperactivation of HFLS induced by TGF-β1, and the effects of oltipraz could be reversed by the exogenous TRAP-1. In short, our research has revealed for the first time that Nrf2/miR338-3p/TRAP-1 pathway was involved in hyperactivation of HFLS in OA patients, Nrf2 has the potential to be used as therapy and new drug target of OA.
PubMed: 37920489
DOI: 10.1016/j.heliyon.2023.e21412