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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Article in English | MEDLINE | ID: covidwho-1662678

ABSTRACT

Cordyline terminalis leaf extract (aqCT) possesses abundant polyphenols and other bioactive compounds, which are encapsulated in gelatin-polyethylene glycol-tyramine (GPT)/alpha-cyclodextrin (α-CD) gels to form the additional functional materials for biomedical applications. In this study, the gel compositions are optimized, and the GPT/α-CD ratios equal to or less than one half for solidification are found. The gelation time varies from 40.7 min to 5.0 h depending on the increase in GPT/α-CD ratios and aqCT amount. The aqCT extract disturbs the hydrogen bonding and host-guest inclusion of GPT/α-CD gel networks, postponing the gelation. Scanning electron microscope observation shows that all gels with or without aqCT possess a microarchitecture and porosity. GPT/α-CD/aqCT gels could release polyphenols from 110 to 350 nmol/mL at the first hour and sustainably from 5.5 to 20.2 nmol/mL for the following hours, which is controlled by feeding the aqCT amount and gel properties. GPT/α-CD/aqCT gels achieved significant antioxidant activity through a 100% scavenging DPPH radical. In addition, all gels are non-cytotoxic with a cell viability more than 85%. Especially, the GPT3.75α-CD10.5aqCT gels with aqCT amount of 3.1-12.5 mg/mL immensely enhanced the cell proliferation of GPT3.75α-CD10.5 gel without extract. These results suggest that the inherent bioactivities of aqCT endowed the resulting GPT/α-CD/aqCT gels with effective antioxidant and high biocompatibility, and natural polyphenols sustainably release a unique platform for a drug delivery system or other biomedical applications.


Subject(s)
Cordyline/chemistry , Dermis/drug effects , Fibroblasts/drug effects , Gels/chemistry , Plant Extracts/pharmacology , Plant Leaves/chemistry , Polyphenols/pharmacology , Cells, Cultured , Drug Liberation , Gels/administration & dosage , Humans
2.
Mar Drugs ; 19(10)2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1480860

ABSTRACT

To discover the new medical entity from edible marine algae, our continuously natural product investigation focused on endophytes from marine macroalgae Grateloupia sp. Two new azaphilones, 8a-epi-hypocrellone A (1), 8a-epi-eupenicilazaphilone C (2), together with five known azaphilones, hypocrellone A (3), eupenicilazaphilone C (4), ((1E,3E)-3,5-dimethylhepta-1,3-dien-1-yl)-2,4-dihydroxy-3-methylbenzaldehyde (5), sclerotiorin (6), and isochromophilone IV (7) were isolated from the alga-derived fungus Penicillium sclerotiorum. The structures of isolated azaphilones (1-7) were elucidated by spectrometric identification, especially HRESIMS, CD, and NMR data analyses. Concerning bioactivity, cytotoxic, anti-inflammatory, and anti-fibrosis activities of those isolates were evaluated. As a result, compound 1 showed selective toxicity toward neuroblastoma cell line SH-SY5Y among seven cancer and one fibroblast cell lines. 20 µM of compounds 1, 3, and 7 inhibited the TNF-α-induced NFκB phosphorylation but did not change the NFκB activity. Compounds 2 and 6 respectively promoted and inhibited SMAD-mediated transcriptional activities stimulated by TGF-ß.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/pharmacology , Benzopyrans/pharmacology , Microalgae , Penicillium , Pigments, Biological/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Aquatic Organisms , Benzopyrans/chemistry , Benzopyrans/therapeutic use , Cell Line, Tumor/drug effects , Fibroblasts/drug effects , Functional Food , Neuroblastoma/drug therapy , Pigments, Biological/chemistry , Pigments, Biological/therapeutic use , Structure-Activity Relationship
3.
J Ethnopharmacol ; 283: 114701, 2022 Jan 30.
Article in English | MEDLINE | ID: covidwho-1446835

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu Decoction (XFBD), one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, plays an important role in the treatment of mild and/or common patients with dampness-toxin obstructing lung syndrome. AIM OF THE STUDY: The present work aims to elucidate the protective effects and the possible mechanism of XFBD against the acute inflammation and pulmonary fibrosis. METHODS: We use TGF-ß1 induced fibroblast activation model and LPS/IL-4 induced macrophage inflammation model as in vitro cell models. The mice model of lung fibrosis was induced by BLM via endotracheal drip, and then XFBD (4.6 g/kg, 9.2 g/kg) were administered orally respectively. The efficacy and molecular mechanisms in the presence or absence of XFBD were investigated. RESULTS: The results proved that XFBD can effectively inhibit fibroblast collagen deposition, down-regulate the level of α-SMA and inhibit the migration of fibroblasts. IL-4 induced macrophage polarization was also inhibited and the secretions of the inflammatory factors including IL6, iNOS were down-regulated. In vivo experiments, the results proved that XFBD improved the weight loss and survival rate of the mice. The XFBD high-dose administration group had a significant effect in inhibiting collagen deposition and the expression of α-SMA in the lungs of mice. XFBD can reduce bleomycin-induced pulmonary fibrosis by inhibiting IL-6/STAT3 activation and related macrophage infiltration. CONCLUSIONS: Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.


Subject(s)
COVID-19/drug therapy , Drugs, Chinese Herbal , Inflammation/drug therapy , Macrophages/drug effects , SARS-CoV-2 , Signal Transduction/drug effects , Animals , Cell Survival/drug effects , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Gene Regulatory Networks , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Phytotherapy , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/prevention & control , RAW 264.7 Cells , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism
4.
Molecules ; 26(17)2021 Aug 27.
Article in English | MEDLINE | ID: covidwho-1403854

ABSTRACT

This paper presents the results of the first part of testing a novel electrospun fiber mat based on a unique macromolecule: polyisobutylene (PIB). A PIB-based compound containing zinc oxide (ZnO) was electrospun into self-supporting mats of 203.75 and 295.5 g/m2 that were investigated using a variety of techniques. The results show that the hydrophobic mats are not cytotoxic, resist fibroblast cell adhesion and biofilm formation and are comfortable and easy to breathe through for use as a mask. The mats show great promise for personal protective equipment and other applications.


Subject(s)
Polyenes/chemistry , Polymers/chemistry , Biofilms/drug effects , Cell Adhesion/drug effects , Cells, Cultured , Fibroblasts/drug effects , Humans , Materials Testing/methods , Nanofibers/chemistry , Zinc Oxide/chemistry
5.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Article in English | MEDLINE | ID: covidwho-1373615

ABSTRACT

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Subject(s)
Antibodies, Neutralizing/pharmacology , Containment of Biohazards/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Viral Matrix Proteins/antagonists & inhibitors , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Embryo, Mammalian , Fibroblasts/drug effects , Fibroblasts/virology , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , MCF-7 Cells , Mice , Molecular Mimicry , Plasmids/chemistry , Plasmids/metabolism , Primary Cell Culture , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Transfection , Vero Cells , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism
6.
DNA Repair (Amst) ; 106: 103180, 2021 10.
Article in English | MEDLINE | ID: covidwho-1313048

ABSTRACT

Since the early stages of the pandemic, hydroxychloroquine (HCQ), a widely used drug with good safety profile in clinic, has come to the forefront of research on drug repurposing for COVID-19 treatment/prevention. Despite the decades-long use of HCQ in the treatment of diseases, such as malaria and autoimmune disorders, the exact mechanisms of action of this drug are only beginning to be understood. To date, no data are available on the genotoxic potential of HCQ in vitro or in vivo. The present study is the first investigation of the DNA damaging- and mutagenic effects of HCQ in mammalian cells in vitro, at concentrations that are comparable to clinically achievable doses in patient populations. We demonstrate significant induction of a representative oxidative DNA damage (8-oxodG) in primary mouse embryonic fibroblasts (MEFs) treated with HCQ at 5 and 25 µM concentrations (P = 0.020 and P = 0.029, respectively), as determined by enzyme-linked immunosorbent assay. Furthermore, we show significant mutagenicity of HCQ, manifest as 2.2- and 1.8-fold increases in relative cII mutant frequency in primary and spontaneously immortalized Big Blue® MEFs, respectively, treated with 25 µM dose of this drug (P = 0.005 and P = 0.012, respectively). The observed genotoxic effects of HCQ in vitro, achievable at clinically relevant doses, are novel and important, and may have significant implications for safety monitoring in patient populations. Given the substantial number of the world's population receiving HCQ for the treatment of various chronic diseases or in the context of clinical trials for COVID-19, our findings warrant further investigations into the biological consequences of therapeutic/preventive use of this drug.


Subject(s)
Hydroxychloroquine/pharmacology , Mutation/drug effects , Oxidative Stress/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Fibroblasts/drug effects , Fibroblasts/virology , Mammals/virology , Mice , Mice, Inbred C57BL , Pandemics/prevention & control , SARS-CoV-2/drug effects
7.
Eur J Immunol ; 51(9): 2330-2340, 2021 09.
Article in English | MEDLINE | ID: covidwho-1261763

ABSTRACT

The molecular mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein was characterized to identify novel therapies. The impact of tofacitinib, IL-6R Ab, or TNFi therapy was determined on Spike protein or LPS/IFN-γ-induced signaling, inflammation, and metabolic reprogramming in MΦs and/or rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS). ACE2 frequency was markedly expanded in MΦs compared to T cells and RA FLS. Tofacitinib suppresses Spike protein potentiated STAT1 signaling, whereas this function was unchanged by TNFi. Tofacitinib impairs IL-6/IFN/LPS-induced STAT1 and STAT3 phosphorylation in RA MΦs and FLS. Interestingly, tofacitinib had a broader inhibitory effect on the monokines, glycolytic regulators, or oxidative metabolites compared to IL-6R Ab and TNFi in Spike-protein-activated MΦs. In contrast, all three therapies disrupted IFN-α and IFN-ß secretion in response to Spike protein; nonetheless, the IFN-γ was only curtailed by tofacitinib or IL-6R Ab. While tofacitinib counteracted MΦ metabolic rewiring instigated by Spike protein, it was inconsequential on the glycolysis expansion mediated via HK2 and/or LDHA in the activated RA MΦ and FLS. Nevertheless, the potentiated inflammatory response and the diminished oxidative phosphorylation modulated by Spike protein and/or LPS/IFN-γ stimulation in MΦs or RA FLS were reversed by tofacitinib. In conclusion, tofacitinib suppresses MΦ inflammation and immunometabolism triggered by Spike protein and may provide a promising strategy for COVID-19 patients.


Subject(s)
COVID-19/drug therapy , Macrophages/drug effects , Piperidines/pharmacology , Pyrimidines/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Arthritis, Rheumatoid/metabolism , COVID-19/metabolism , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Interleukin-6/metabolism , Macrophages/metabolism , Receptors, Interleukin-6/metabolism , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/metabolism
8.
SLAS Discov ; 26(9): 1091-1106, 2021 10.
Article in English | MEDLINE | ID: covidwho-1255878

ABSTRACT

Lung imaging and autopsy reports among COVID-19 patients show elevated lung scarring (fibrosis). Early data from COVID-19 patients as well as previous studies from severe acute respiratory syndrome, Middle East respiratory syndrome, and other respiratory disorders show that the extent of lung fibrosis is associated with a higher mortality, prolonged ventilator dependence, and poorer long-term health prognosis. Current treatments to halt or reverse lung fibrosis are limited; thus, the rapid development of effective antifibrotic therapies is a major global medical need that will continue far beyond the current COVID-19 pandemic. Reproducible fibrosis screening assays with high signal-to-noise ratios and disease-relevant readouts such as extracellular matrix (ECM) deposition (the hallmark of fibrosis) are integral to any antifibrotic therapeutic development. Therefore, we have established an automated high-throughput and high-content primary screening assay measuring transforming growth factor-ß (TGFß)-induced ECM deposition from primary human lung fibroblasts in a 384-well format. This assay combines longitudinal live cell imaging with multiparametric high-content analysis of ECM deposition. Using this assay, we have screened a library of 2743 small molecules representing approved drugs and late-stage clinical candidates. Confirmed hits were subsequently profiled through a suite of secondary lung fibroblast phenotypic screening assays quantifying cell differentiation, proliferation, migration, and apoptosis. In silico target prediction and pathway network analysis were applied to the confirmed hits. We anticipate this suite of assays and data analysis tools will aid the identification of new treatments to mitigate against lung fibrosis associated with COVID-19 and other fibrotic diseases.


Subject(s)
COVID-19/drug therapy , Drug Discovery , Lung/diagnostic imaging , Small Molecule Libraries/pharmacology , Apoptosis/drug effects , COVID-19/epidemiology , COVID-19/virology , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/pathology , Fibroblasts/drug effects , Humans , Lung/drug effects , Lung/pathology , Lung/virology , Mass Screening , Pandemics , SARS-CoV-2/pathogenicity , Signal Transduction/drug effects
10.
Lett Appl Microbiol ; 72(6): 725-729, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1096912

ABSTRACT

There does not appear to be any studies in the published literature on the stability of SARS-CoV-2 in climbing chalk powder (magnesium carbonate and/or calcium carbonate), which has been hypothesized to pose a potential risk of fomite transmission of coronavirus disease 2019 (COVID-19) within climbing gyms. The aim of this study was to determine the infectivity of a model human coronavirus HCoV-OC43 in the presence of climbing chalk powder on a dry plastic surface. The stability of HCoV-OC43 on a plastic surface dusted with climbing chalk powders (magnesium carbonate, calcium carbonate or a blended chalk) was determined by titration on BHK-21 fibroblast cells. No chalk and no virus controls were included. HCoV-OC43 was stable on the plastic surface for 48 h. The stability of HCoV-OC43 was significantly (P ≤ 0·05) reduced in the presence of magnesium carbonate, calcium carbonate and the chalk blend; the infectivity was reduced by ≥2·29 log10 50% tissue culture infective dose (TCID50 ) immediately upon on contact and by ≥2·46 log10 TCID50 within 1 h of contact. These findings suggest that the infectivity of coronaviruses is reduced by climbing chalk, limiting the risk of potential fomite transmission.


Subject(s)
Coronavirus OC43, Human/drug effects , Animals , Calcium Carbonate , Cell Line , Coronavirus OC43, Human/pathogenicity , Cricetinae , Fibroblasts/drug effects , Fibroblasts/virology , Fomites , Powders
11.
Aging (Albany NY) ; 13(2): 1571-1590, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-1040203

ABSTRACT

The main aspects of severe COVID-19 disease pathogenesis include hyper-induction of proinflammatory cytokines, also known as 'cytokine storm', that precedes acute respiratory distress syndrome (ARDS) and often leads to death. COVID-19 patients often suffer from lung fibrosis, a serious and untreatable condition. There remains no effective treatment for these complications. Out of all cytokines, TNFα and IL-6 play crucial roles in cytokine storm pathogenesis and are likely responsible for the escalation in disease severity. These cytokines also partake in the molecular pathogenesis of fibrosis. Therefore, new approaches are urgently needed, that can efficiently and swiftly downregulate TNFα, IL-6, and the inflammatory cytokine cascade, in order to curb inflammation and prevent fibrosis, and lead to disease remission. Cannabis sativa has been proposed to modulate gene expression and inflammation and is under investigation for several potential therapeutic applications against autoinflammatory diseases and cancer. Here, we hypothesized that the extracts of novel C. sativa cultivars may be used to downregulate the expression of pro-inflammatory cytokines and pathways involved in inflammation and fibrosis. Initially, to analyze the anti-inflammatory effects of novel C. sativa cultivars, we used a well-established full thickness human 3D skin artificial EpiDermFTTM tissue model, whereby tissues were exposed to UV to induce inflammation and then treated with extracts of seven new cannabis cultivars. We noted that out of seven studied extracts of novel C. sativa cultivars, three (#4, #8 and #14) were the most effective, causing profound and concerted down-regulation of COX2, TNFα, IL-6, CCL2, and other cytokines and pathways related to inflammation and fibrosis. These data were further confirmed in the WI-38 lung fibroblast cell line model. Most importantly, one of the tested extracts had no effect at all, and one exerted effect that may be deleterious, signifying that careful cannabis cultivar selection must be based on thorough pre-clinical studies. The observed pronounced inhibition of TNFα and IL-6 is the most important finding, because these molecules are currently considered to be the main targets in COVID-19 cytokine storm and ARDS pathogenesis. Novel anti-TNFα and anti-IL-6 cannabis extracts can be useful additions to the current anti-inflammatory regimens to treat COVID-19, as well as various rheumatological diseases and conditions, and 'inflammaging' - the inflammatory underpinning of aging and frailty.


Subject(s)
COVID-19 , Cannabis , Cytokine Release Syndrome , Interleukin-6/antagonists & inhibitors , Plant Extracts/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , COVID-19/complications , Cannabinoids/pharmacology , Cell Line , Fibroblasts/drug effects , Humans , Inflammation/virology , SARS-CoV-2 , Skin/drug effects , Tissue Culture Techniques
12.
Int J Mol Sci ; 21(22)2020 Nov 10.
Article in English | MEDLINE | ID: covidwho-917002

ABSTRACT

Pro-inflammatory cytokines like interleukin-1ß (IL-1ß) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1ß on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1ß and used Atomic Force Microscopy to unveil that IL-1ß significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1ß stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1ß may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1ß-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1ß provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.


Subject(s)
Interleukin-1beta/physiology , Lung/physiology , Actins/metabolism , Adolescent , Adult , Biomechanical Phenomena , Cell Movement/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type III/genetics , Collagen Type III/metabolism , Cyclooxygenase 2/metabolism , Elasticity/drug effects , Elasticity/physiology , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , Interleukin-1beta/pharmacology , Lung/cytology , Lung/drug effects , Male , Microscopy, Atomic Force , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regeneration/genetics , Regeneration/physiology , Wound Healing/drug effects , Wound Healing/genetics , Wound Healing/physiology , Young Adult
13.
Int J Mol Sci ; 21(23)2020 Dec 03.
Article in English | MEDLINE | ID: covidwho-965280

ABSTRACT

Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients' ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR's influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts.


Subject(s)
Fibroblasts/drug effects , Fibroblasts/metabolism , Glucocorticoids/pharmacology , Receptors, Glucocorticoid/metabolism , Transcription Factors/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Fluorescent Antibody Technique , Humans , Hydrocortisone/pharmacology , Methylprednisolone/pharmacology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/metabolism , Protein Binding
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