RESUMO
ChIP-qPCR offers the opportunity to identify interactions of DNA-binding proteins such as transcription factors and their respective DNA binding sites. Thereby, transcription factors can interfere with gene expression, resulting in up- or downregulation of their target genes. Utilizing ChIP, it is possible to identify specific DNA binding sites that are bound by the DNA-binding proteins in dependence on treatment or prevailing conditions. During ChIP, DNA-binding proteins are reversibly cross-linked to their DNA binding sites and the DNA itself is fragmented. Using bead-captured antibodies, the target proteins are isolated while still binding their respective DNA response element. Using quantitative PCR, these DNA fragments are amplified and quantified. In this protocol, DNA binding sites of the glucocorticoid receptor are identified by treatment with the synthetic glucocorticoid Dexamethasone in murine bone marrow-derived macrophages.
Assuntos
Imunoprecipitação da Cromatina , Receptores de Glucocorticoides , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Animais , Imunoprecipitação da Cromatina/métodos , Camundongos , Sítios de Ligação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ligação Proteica , Dexametasona/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , DNA/metabolismo , DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.
Assuntos
Imunoprecipitação da Cromatina , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos , Receptores de Glucocorticoides , Animais , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Camundongos , Macrófagos/metabolismo , DNA/metabolismo , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação da Cromatina/métodos , Ligação Proteica , Sítios de LigaçãoRESUMO
Paradoxical sleep deprivation (PSD) presents different effects on metabolism and neurological functions. In addition, over long duration, sleep restriction (SR) can promote permanent changes. The prostate is an endocrine-dependent organ with homeostatic regulation directly related to hormone levels. Our study proposed to demonstrate the experimental prostatic effects of PSD (96 h), PSD with recovery (PSR - 96/96 h), and sleep restriction (SR - 30 PSD cycles/recovery). PSD and SR promoted decrease in serum testosterone and significant increase in serum and intraprostatic corticosterone. In agreement, androgen receptors (AR) were less expressed and glucocorticoid receptors (GR) were enhanced in PSR and SR. Thus, the prostate, especially under SR, demonstrates a castration-like effect due to loss of responsiveness and sensitization by androgens. SR triggered an important inflammatory response through enhancement of serum and intraprostatic pro- (IL-1α, IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines. Furthermore, the respective receptors of anti-inflammatory cytokines (IL-1RI and TNF-R) were highly expressed in the prostatic epithelium and stroma. PSR can partially restore prostate homeostasis, as it restores testosterone and the prostate proliferation index, in addition to promoting balance in the inflammatory response that is considered protective. PSD and SR are key factors in the endocrine axis that coordinate prostatic homeostasis, and significant changes in these factors have consequences on prostate functionality.
Assuntos
Gerbillinae , Próstata , Receptores Androgênicos , Privação do Sono , Testosterona , Animais , Masculino , Privação do Sono/metabolismo , Privação do Sono/patologia , Próstata/metabolismo , Próstata/patologia , Testosterona/sangue , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Corticosterona/sangue , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Castração , Androgênios/metabolismoRESUMO
OBJECTIVE: This study was designed to determine the comparative efficacy of Doxofylline (DOXO) compared to low-dose theophylline (LDT) in treating corticosteroid-resistant asthma. METHODS: This study was conducted on 56 adult BALB/C mice aged six to eight weeks old with an average weight of 20-25 g. They were divided into seven groups: control group, ovalbumin (OVA)+lipopolysaccharide (LPS) group, OVA+LPS+dexamethasone (DEXA) group, OVA+LPS+LDT group, OVA+LPS+ group, OVA+LPS+DEXA+LDT group, and OVA +LPS+DEXA+DOXO group. All mice were administered IP DOXO+DEXA. All the doses were administrated one day before the first challenge and lasted for five consecutive days after one hour of the OVA challenge until sacrificed. Lung biochemical parameters, including interleukin (IL)-2, IL-4, IL-8, IL-10, and IL-17 levels, were measured using enzyme-linked immunosorbent assay (ELISA). In addition, Histone deacetylase (HDAC) activity and lung histological analysis were also performed. Furthermore, the glucocorticoid receptor was measured by nexttec™. RESULTS: The OVA+LPS group exhibited significantly (p<0.05) elevated levels of interleukin (IL)-2, IL-4, IL-8, IL-10, and IL-17 compared to controls, indicative of airway inflammation. Moreover, OVA+LPS induction significantly (p<0.05) increased the levels of Interferon-gamma (IFN-γ), NF[Formula: see text]B, Tumor Necrosis Factor (TNFα), and Immunoglobulin E (IgE) parameters, indicating severe inflammation and immune response and successfully induced the disease model. Meanwhile, LDT and DOXO in conjunction with DEXA, further augmented HDAC2 activity compared to DEXA alone. Similarly, the administration of LDT increased the expression of GR by 64.5% (23.72±0.34), while DOXO increased the expression of GR by 94.10% (27.99±0.15), which restores it back to control. Furthermore, according to Hematoxylin and eosin (H&E) stained sections, the DOXO group exhibited a slight improvement in these histopathological features, suggesting a modest therapeutic effect. Masson's Trichrome staining showed a slightly improved patchy collagen deposition within alveolar spaces in intra-alveolar and interstitial inflammatory cell accumulation in DOXO group, and the combination of these drugs (DEXA+LDT group) improved collagen deposition moderately within alveolar spaces in intra-alveolar and interstitial inflammatory cell accumulation. Overall, treatment with DOXO, LDT alone, and with DEXA combination led to reductions in cytokine levels, with DOXO and LDT showing significant (p<0.05) efficacy to DEXA used alone, which showed non-significant (p>0.05) efficacy. CONCLUSIONS: Doxofylline and LDT were found to be effective therapeutic agents when used alone or in combination with Dexamethasone. However, randomized controlled trials are required to evaluate its further efficacy.
Assuntos
Asma , Dexametasona , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Teofilina , Animais , Teofilina/farmacologia , Teofilina/análogos & derivados , Teofilina/administração & dosagem , Asma/tratamento farmacológico , Asma/patologia , Dexametasona/farmacologia , Dexametasona/administração & dosagem , Camundongos , Corticosteroides/farmacologia , Corticosteroides/uso terapêutico , Citocinas/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismo , Ovalbumina , Receptores de Glucocorticoides/metabolismo , Histona Desacetilases/metabolismo , Lipopolissacarídeos/farmacologiaRESUMO
Glucocorticoidsï¼GCï¼ are widely used in the clinical treatment of autoimmune inner ear diseases, sudden sensorineural hearing loss, Meniere's disease, sinusitis and other otolaryngology diseases. However, GC resistance remains a major factor contributing to the poor efficacy of clinical treatments. The mechanism of GC resistance is still unclear. This paper reviews the related mechanisms of GC resistance from the perspectives of GC receptor factors and non-GC receptor factors. Additionally, it summarizes the latest research progress on GC resistance in otolaryngological diseases, with the aim of identifying effective clinical alternative treatment options for reversing GC resistance in the future.
Assuntos
Resistência a Medicamentos , Glucocorticoides , Otorrinolaringopatias , Receptores de Glucocorticoides , Humanos , Glucocorticoides/uso terapêutico , Otorrinolaringopatias/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Doença de Meniere/tratamento farmacológicoRESUMO
Fear conditioning evokes a physiologic release of glucocorticoids that assists learning. As a cochaperone in the glucocorticoid receptor complex, FKBP51 modulates stress-induced glucocorticoid signaling and may influence conditioned fear responses. This study combines molecular and behavioral approaches to examine whether locally reducing FKBP51 expression in the ventral hippocampus is sufficient to affect fear-related behaviors. We hypothesized that reducing FKBP51 expression in the VH would increase glucocorticoid signaling to alter auditory fear conditioning. Adult male rats were injected with an adeno-associated virus (AAV) vector expressing short hairpin - RNAs (shRNA) targeting FKBP5 into the ventral hippocampus to reduce FKBP5 levels or a control AAV. Infusion of FKBP5-shRNA into the ventral hippocampus decreased auditory fear acquisition and recall. Although animals injected with FKBP5-shRNA showed less freezing during extinction recall, the difference was due to a reduced fear recall rather than improved extinction. Reducing ventral hippocampus FKBP51 did not affect exploratory behavior in either the open field test or the elevated zero maze test but did increase passive behavior in the forced swim test, suggesting that the reduction in auditory fear recall was not due to more active responses to acute stress. Furthermore, lower ventral hippocampus FKBP51 levels did not alter corticosterone release in response to restraint stress, suggesting that the reduced fear recall was not due to lower corticosterone release. Our findings suggest FKBP51 in the ventral hippocampus plays a selective role in modulating fear-learning processes and passive behavioral responses to acute stress rather than hypothalamic-pituitary-adrenal axis reactivity or exploratory responses.
Assuntos
Medo , Hipocampo , Proteínas de Ligação a Tacrolimo , Animais , Masculino , Medo/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Hipocampo/metabolismo , Ratos , Corticosterona/metabolismo , Corticosterona/sangue , Ratos Sprague-Dawley , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Receptores de Glucocorticoides/metabolismo , Extinção Psicológica/fisiologiaRESUMO
Cortisol is a clinically validated stress biomarker that takes part in many physiological and psychological functions related to the body's response to stress factors. In particular, it has emerged as a pivotal tool for understanding stress levels and overall well-being. Usually, in clinics, cortisol levels are monitored in blood or urine, but significant changes are also registered in sweat and saliva. In this work, a surface plasmon resonance probe based on a D-shaped plastic optical fiber was functionalized with a glucocorticoid receptor exploited as a highly efficient bioreceptor specific to cortisol. The developed plastic optical fiber biosensor was tested for cortisol detection in buffer and artificial saliva. The biosensor response showed very good selectivity towards other hormones and a detection limit of about 59 fM and 96 fM in phosphate saline buffer and artificial saliva, respectively. The obtained detection limit, with a rapid detection time (about 5 min) and a low-cost sensor system, paved the way for determining the cortisol concentration in saliva samples without any extraction process or sample pretreatment via a point-of-care test.
Assuntos
Técnicas Biossensoriais , Hidrocortisona , Fibras Ópticas , Saliva , Ressonância de Plasmônio de Superfície , Hidrocortisona/análise , Saliva/química , Humanos , Limite de Detecção , Plásticos , Receptores de GlucocorticoidesRESUMO
In adult rats, omega-3 supplementation through fish oil (FO) and environmental enrichment (EE) have shown beneficial effects on cognition and stress regulation. This study assessed sex-specific effects of FO and EE during adolescence, a period critical for brain maturation, on adulthood coping mechanisms, sociability, and glucocorticoid regulation. An amount of 64 Wistar rats [n = 32/sex; postnatal day (PND) 23] were assigned to supplementation of control soybean oil (CSO) or menhaden fish oil (FO; 0.3 mL/100 g) from PND28 to 47 and exposed to EE or regular cage (RC) housing from PND28 to 58, with their blood corticosterone (CORT) levels being assessed weekly. As adults, exposure to repeated forced swim tests (FSTs; PND90-91) enabled analysis of coping responses, while socioemotional and memory responses were evaluated using the OFT, EPM, SIT, and Y maze tests (PND92-94). Immunohistochemistry determined hippocampal CA1/CA3 glucocorticoid receptor (GR) expression (PND95). CORT secretion gradually increased as the supplementation period elapsed in female rats, while changes were minimal in males. Coping strategies in the FST differed between sexes, particularly in FO-fed rats, where females and males, respectively, favoured floating and tail support to minimise energy consumption and maintain immobility. In the SIT, FO/EE promoted sociability in females, while a CSO diet favoured social recognition in males. Reduced CA3 GR-ir expression was found in FO/RC and CSO/EE rat groups, supporting stress resilience and memory consolidation. Our findings support environment and dietary conditions to exert a sex-specific impact on biobehavioural responses.
Assuntos
Adaptação Psicológica , Corticosterona , Ácidos Graxos Ômega-3 , Ratos Wistar , Receptores de Glucocorticoides , Estresse Psicológico , Animais , Receptores de Glucocorticoides/metabolismo , Masculino , Feminino , Corticosterona/sangue , Ácidos Graxos Ômega-3/farmacologia , Estresse Psicológico/metabolismo , Ratos , Suplementos Nutricionais , Meio Ambiente , Comportamento Social , Comportamento Animal , Região CA3 Hipocampal/metabolismo , Óleos de Peixe/farmacologia , Óleos de Peixe/administração & dosagem , Fatores SexuaisRESUMO
BACKGROUND: Stress during pregnancy can lead to adverse maternal and infant health outcomes through epigenetic changes in the hypothalamic-pituitary-adrenal axis. Among farmers in low-income countries, one important stressor is food insecurity, which can be reduced using hermetic storage bags. This study aimed to determine, for the first time, whether a hermetic storage bag intervention during pregnancy positively affects maternal and infant DNA methylation of the hypothalamic-pituitary-adrenal axis-related genes FKBP5 and NR3C1. We further analyzed whether anthropometrics, stress, and mental health were associated with DNA methylation. METHODS: This study was part of a larger matched-pair randomized controlled trial focusing on the impact of improved on-farm storage on food security, poverty, and net income of smallholder farming households. A total of N = 149 mothers were recruited by telephone and invited to attend a study appointment at health facilities in Kakamega County, Western Kenya, with their infants in April or May 2021. During the appointment, anthropometric measurements were taken, questionnaires on stress and mental health were administered, and saliva samples were collected. Logistic and multiple linear regression were used to examine the effect of the intervention and related measures on DNA methylation. RESULTS: Mothers in the intervention group showed higher mean NR3C1 methylation levels than those in the control group, corrected for multiple testing. Maternal postpartum body mass index was positively associated with infant NR3C1 CpG3 DNA methylation. The more stressful life events a mother had experienced in the previous 12 months (including during pregnancy), the lower her FKBP5 CpG3 methylation levels. CONCLUSIONS: Food insecurity and stressful life events during pregnancy seem to exert significant effects on maternal DNA methylation. While these stressors did not appear to impact infant DNA methylation in the present study, maternal postpartum body mass index was significantly related to infant methylation. These findings suggest that while infants may be protected from excessive maternal glucocorticoids by placental barrier activity, maternal metabolic status is still reflected in their epigenetic make-up. Trial registration This study was part of a larger matched-pair randomized controlled trial on the impact of improved on-farm crop storage on welfare, nutrition, and human health. Registration can be found in the American Economic Association (AEA) RCT Registry, RCT ID: AEARCTR-0005845.
Assuntos
Metilação de DNA , Epigênese Genética , Receptores de Glucocorticoides , Humanos , Metilação de DNA/genética , Feminino , Quênia , Adulto , Gravidez , Lactente , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Mães/psicologia , Masculino , Estresse Psicológico/genética , Fazendas , Sistema Hipotálamo-Hipofisário/metabolismo , Adulto Jovem , Insegurança Alimentar , Sistema Hipófise-Suprarrenal/metabolismo , Recém-Nascido , Produtos Agrícolas/genéticaRESUMO
BACKGROUND: Bariatric surgery is the most effective treatment for severe obesity. There can be variation in the degree of weight reduction following bariatric surgery. It is unknown whether single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor locus (GRL) affect postoperative weight loss and metabolic outcomes. MATERIALS/METHODS: We studied the association between selected candidate SNPs and postoperative weight loss and metabolic outcomes in patients with severe obesity undergoing bariatric surgery. The polymorphisms rs41423247 (Bcl1), rs56149945 (N363S) and rs6189/rs6190 (ER22/23EK) were analysed. RESULTS: The 139 participants included 95 women (68.3%) and had a median (interquartile range) age of 53.0 (46.0-60.0) years and mean (SD) weight of 140.8 (28.8) kg and body mass index of 50.3 (8.6) kg/m2. At baseline, 59 patients had type 2 diabetes (T2D), 60 had hypertension and 35 had obstructive sleep apnoea syndrome treated with continuous positive airway pressure (CPAP). 84 patients (60.4%) underwent gastric bypass and 55 (39.6%) underwent sleeve gastrectomy. There were no significant differences in weight loss, glycated haemoglobin (HbA1c) or lipid profile categorized by genotype status, sex or median age. There was significant weight reduction after bariatric surgery with a postoperative BMI of 34.1 (6.8) kg/m2 at 24 months (p < 0.001). CONCLUSION: While GRL polymorphisms with a known deleterious effect on adipose tissue mass and function may have a small, additive effect on the prevalence of obesity and related metabolic disorders in the population, we suggest that the relatively weak biological influence of these SNPs is readily overcome by bariatric surgery.
Assuntos
Cirurgia Bariátrica , Polimorfismo de Nucleotídeo Único , Receptores de Glucocorticoides , Redução de Peso , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Redução de Peso/genética , Estudos Prospectivos , Resultado do Tratamento , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/cirurgia , Obesidade Mórbida/cirurgia , Obesidade Mórbida/genética , Obesidade Mórbida/metabolismo , AdultoRESUMO
Alpha-1-antitrypsin (AAT) plays a homeostatic role in attenuating excessive inflammation and augmenting host defense against microbes. We demonstrated previously that AAT binds to the glucocorticoid receptor (GR) resulting in significant anti-inflammatory and antimycobacterial consequences in macrophages. Our current investigation aims to uncover AAT-regulated genes that rely on GR in macrophages. We incubated control THP-1 cells (THP-1control) and THP-1 cells knocked down for GR (THP-1GR-KD) with AAT, performed bulk RNA sequencing, and analyzed the findings. In THP-1control cells, AAT significantly upregulated 408 genes and downregulated 376 genes. Comparing THP-1control and THP-1GR-KD cells, 125 (30.6%) of the AAT-upregulated genes and 154 (41.0%) of the AAT-downregulated genes were significantly dependent on GR. Among the AAT-upregulated, GR-dependent genes, CSF-2 that encodes for granulocyte-monocyte colony-stimulating factor (GM-CSF), known to be host-protective against nontuberculous mycobacteria, was strongly upregulated by AAT and dependent on GR. We further quantified the mRNA and protein of several AAT-upregulated, GR-dependent genes in macrophages and the mRNA of several AAT-downregulated, GR-dependent genes. We also discussed the function(s) of selected AAT-regulated, GR-dependent gene products largely in the context of mycobacterial infections. In conclusion, AAT regulated several genes that are dependent on GR and play roles in host immunity against mycobacteria.
Assuntos
Macrófagos , Receptores de Glucocorticoides , alfa 1-Antitripsina , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Humanos , Macrófagos/metabolismo , Macrófagos/imunologia , Células THP-1 , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genéticaRESUMO
Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.
Assuntos
Peptídeos , Proteômica , SARS-CoV-2 , Proteômica/métodos , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Peptídeos/metabolismo , Peptídeos/química , COVID-19/metabolismo , COVID-19/virologia , Células HEK293 , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/química , Plasmídeos/genética , Plasmídeos/metabolismo , Espectrometria de Massas/métodos , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Mapeamento de Interação de Proteínas/métodosRESUMO
Liver fibrosis is a determinant-stage process of many chronic liver diseases and affected over 7.9 billion populations worldwide with increasing demands of ideal therapeutic agents. Discovery of active molecules with anti-hepatic fibrosis efficacies presents the most attacking filed. Here, we revealed that hepatic L-aspartate levels were decreased in CCl4-induced fibrotic mice. Instead, supplementation of L-aspartate orally alleviated typical manifestations of liver injury and fibrosis. These therapeutic efficacies were alongside improvements of mitochondrial adaptive oxidation. Notably, treatment with L-aspartate rebalanced hepatic cholesterol-steroid metabolism and reduced the levels of liver-impairing metabolites, including corticosterone (CORT). Mechanistically, L-aspartate treatment efficiently reversed CORT-mediated glucocorticoid receptor ß (GRß) signaling activation and subsequent transcriptional suppression of the mitochondrial genome by directly binding to the mitochondrial genome. Knockout of GRß ameliorated corticosterone-mediated mitochondrial dysfunction and hepatocyte damage which also weakened the improvements of L-aspartate in suppressing GRß signaling. These data suggest that L-aspartate ameliorates hepatic fibrosis by suppressing GRß signaling via rebalancing cholesterol-steroid metabolism, would be an ideal candidate for clinical liver fibrosis treatment.
Assuntos
Ácido Aspártico , Tetracloreto de Carbono , Cirrose Hepática , Fígado , Camundongos Endogâmicos C57BL , Receptores de Glucocorticoides , Animais , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Masculino , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Ácido Aspártico/metabolismo , Camundongos , Corticosterona , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Colesterol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Camundongos KnockoutRESUMO
Cerebral ischemia-induced systemic inflammation and inflammasome-dependent pyroptotic cell death in ileum, causing serious intestinal injury. Glucocorticoid receptor (GR) mediates the effects of glucocorticoids and participates in inflammation. Escin has corticosteroid-like, neuroprotective, and anti-intestinal dysfunction effects. This study aimed to investigate the effect of Escin on the intestinal barrier injury in rats subjected to middle cerebral artery occlusion (MCAO) and on Caco-2 cells exposed to lipopolysaccharides. The MCAO-caused brain injury was evaluated by assessing neurological function, cerebral infarct volume, and plasma corticosterone (Cort) levels. Intestinal injury was evaluated by observing the histopathological changes, assessing the intestinal barrier function, and determining blood FD4, endotoxin and IL-1ß levels. The levels of the tight-junction proteins such as claudin-1, occludin, and ZO-1, and proteins involved in the GR/p38 MAPK/NF-κB pathway and NLRP3-inflammasome activation were evaluated using western blotting or immunofluorescence. Administration of Escin suppressed the cerebral ischemia-induced increases in Garcia-test scores and infarct volume, alleviated the injury to the intestinal barrier, and decreased the levels of Cort, endotoxin, and IL-1ß. Additionally, Escin upregulated GR and downregulated phospho(p)-p65, p-p38MAPK, NLRP3, GSDMD-N, and cleaved-caspase-1 in the intestine. The effects of Escin could be suppressed by the GR antagonist RU486 or enhanced by the p38 MAPK antagonist SB203580. We revealed details how Escin improves cerebral ischemia-induced intestinal barrier injury by upregulating GR and thereby inhibiting the pyroptosis induced by NF-κB-mediated NLRP3 activation. This study will provide a experimental foundation for the features of glucocorticoid-like activity and the discovery of new clinical application for Escin.
Assuntos
Isquemia Encefálica , Escina , Inflamassomos , Piroptose , Receptores de Glucocorticoides , Transdução de Sinais , Animais , Humanos , Masculino , Ratos , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Células CACO-2 , Modelos Animais de Doenças , Escina/farmacologia , Escina/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Intestinos/patologia , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Lipopolissacarídeos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Piroptose/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder worldwide. Current treatments for PD largely center around dopamine replacement therapies and fail to prevent the progression of pathology, underscoring the need for neuroprotective interventions. Approaches that target neuroinflammation, which occurs prior to dopaminergic neuron (DAn) loss in the substantia nigra (SN), represent a promising therapeutic strategy. The glucocorticoid receptor (GR) has been implicated in the neuropathology of PD and modulates numerous neuroinflammatory signaling pathways in the brain. Therefore, we investigated the neuroprotective effects of the novel GR modulator, PT150, in the rotenone mouse model of PD, postulating that inhibition of glial inflammation would protect DAn and reduce accumulation of neurotoxic misfolded âº-synuclein protein. C57Bl/6 mice were exposed to 2.5â¯mg/kg/day rotenone by intraperitoneal injection for 14 days. Upon completion of rotenone dosing, mice were orally treated at day 15 with 30â¯mg/kg/day or 100â¯mg/kg/day PT150 in the 14-day post-lesioning incubation period, during which the majority of DAn loss and α-synuclein (α-syn) accumulation occurs. Our results indicate that treatment with PT150 reduced both loss of DAn and microgliosis in the nigrostriatal pathway. Although morphologic features of astrogliosis were not attenuated, PT150 treatment promoted potentially neuroprotective activity in these cells, including increased phagocytosis of hyperphosphorylated α-syn. Ultimately, PT150 treatment reduced the loss of DAn cell bodies in the SN, but not the striatum, and prohibited intra-neuronal accumulation of α-syn. Together, these data indicate that PT150 effectively reduced SN pathology in the rotenone mouse model of PD.
Assuntos
Neurônios Dopaminérgicos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores , Receptores de Glucocorticoides , Rotenona , alfa-Sinucleína , Animais , Rotenona/toxicidade , Fármacos Neuroprotetores/farmacologia , Camundongos , Masculino , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/metabolismo , Receptores de Glucocorticoides/metabolismo , alfa-Sinucleína/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Substância Negra/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Modelos Animais de Doenças , FenantrenosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Euonymus alatus (Thunb.) Siebold. (EA), a traditional Chinese medicine, is widely used in the treatment of diabetes. Our group has previously found that EA could treat diabetic retinopathy (DR) and stigmast-4-en-3-one (Numbered E6) is the active substance responsible for inhibiting angiogenesis in vitro by EA. However, the effects and mechanisms of E6 in the treatment of DR is still unknown. AIM OF THE STUDY: The aim of this study was to investigate the effects and mechanisms of E6 in EA on DR. Additionally, a comparison was made between the effects of E6 and triamcinolone acetonide (TA), as well as the side effects of E6 and dexamethasone. MATERIALS AND METHODS: Ocular affinity assessment and pharmacokinetic parameter prediction were conducted to evaluate the potential of E6 to treat DR. Retinal endothelial cells were used to investigate the in vitro inhibitory effect of E6 on vascular proliferation. Additionally, chicken embryos, zebrafish, and mice were used to investigate the in vivo anti-vascular proliferation effect of E6. Finally, diabetic mice were used to investigate whether E6 improves diabetic retinopathy and to compare its efficacy with that of TA. We then used network pharmacology to study the targets of E6 and performed molecular docking; followed by immunofluorescence experiments, ELISA, Western blot, and tube formation experiments to further investigate its mechanism. Finally, we compared the side effects of E6 with those of dexamethasone. RESULTS: E6 was found to have an affinity for the eye and to inhibit vascular proliferation both in vivo and in vitro. Moreover, E6 was found to be more efficacious than TA in the treatment of DR. Molecular docking experiments predicted that the glucocorticoid receptor (GR) is a potential target of E6, and immunofluorescence analyses confirmed that E6 upregulated the expression of the GR in the retina of hyperglycemic mice. In addition, western blotting results and tube formation experiments showed that E6 also attenuated angiogenesis by inhibiting the Hippo and VEGF pathways. Finally, by comparing the effects of E6 and dexamethasone on glucose regulation and osteoporosis, E6 was found to have fewer side effects. CONCLUSIONS: E6 is a highly effective drug for the treatment of DR, superior to TA and with fewer side effects than dexamethasone. Its mechanism involves the activation of glucocorticoid receptor and inhibition of Hippo and VEGF pathways to alleviate angiogenesis and inflammation. This study is the first to investigate the role and mechanism of E6 in improving DR. The findings suggest that E6 has unique advantages in the treatment of DR.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Euonymus , Receptores de Glucocorticoides , Peixe-Zebra , Animais , Retinopatia Diabética/tratamento farmacológico , Camundongos , Receptores de Glucocorticoides/metabolismo , Embrião de Galinha , Diabetes Mellitus Experimental/tratamento farmacológico , Euonymus/química , Masculino , Simulação de Acoplamento Molecular , Dexametasona/farmacologia , Camundongos Endogâmicos C57BL , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores da Angiogênese/farmacologia , Triancinolona Acetonida/farmacologia , AngiogêneseRESUMO
Computational exploration of chemical space is crucial in modern cheminformatics research for accelerating the discovery of new biologically active compounds. In this study, we present a detailed analysis of the chemical library of potential glucocorticoid receptor (GR) ligands generated by the molecular generator, Molpher. To generate the targeted GR library and construct the classification models, structures from the ChEMBL database as well as from the internal IMG library, which was experimentally screened for biological activity in the primary luciferase reporter cell assay, were utilized. The composition of the targeted GR ligand library was compared with a reference library that randomly samples chemical space. A random forest model was used to determine the biological activity of ligands, incorporating its applicability domain using conformal prediction. It was demonstrated that the GR library is significantly enriched with GR ligands compared to the random library. Furthermore, a prospective analysis demonstrated that Molpher successfully designed compounds, which were subsequently experimentally confirmed to be active on the GR. A collection of 34 potential new GR ligands was also identified. Moreover, an important contribution of this study is the establishment of a comprehensive workflow for evaluating computationally generated ligands, particularly those with potential activity against targets that are challenging to dock.
Assuntos
Receptores de Glucocorticoides , Bibliotecas de Moléculas Pequenas , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/química , Ligantes , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , HumanosRESUMO
The association between sleep and the immune-endocrine system is well recognized, but the nature of that relationship is not well understood. Sleep fragmentation induces a pro-inflammatory response in peripheral tissues and brain, but it also activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing glucocorticoids (GCs) (cortisol in humans and corticosterone in mice). It is unclear whether this rapid release of glucocorticoids acts to potentiate or dampen the inflammatory response in the short term. The purpose of this study was to determine whether blocking or suppressing glucocorticoid activity will affect the inflammatory response from acute sleep fragmentation (ASF). Male C57BL/6J mice were injected i.p. with either 0.9% NaCl (vehicle 1), metyrapone (a glucocorticoid synthesis inhibitor, dissolved in vehicle 1), 2% ethanol in polyethylene glycol (vehicle 2), or mifepristone (a glucocorticoid receptor antagonist, dissolved in vehicle 2) 10 min before the start of ASF or no sleep fragmentation (NSF). After 24 h, samples were collected from brain (prefrontal cortex, hypothalamus, hippocampus) and periphery (liver, spleen, heart, and epididymal white adipose tissue (EWAT)). Proinflammatory gene expression (TNF-α and IL-1ß) was measured, followed by gene expression analysis. Metyrapone treatment affected pro-inflammatory cytokine gene expression during ASF in some peripheral tissues, but not in the brain. More specifically, metyrapone treatment suppressed IL-1ß expression in EWAT during ASF, which implies a pro-inflammatory effect of GCs. However, in cardiac tissue, metyrapone treatment increased TNF-α expression in ASF mice, suggesting an anti-inflammatory effect of GCs. Mifepristone treatment yielded more significant results than metyrapone, reducing TNF-α expression in liver (only NSF mice) and cardiac tissue during ASF, indicating a pro-inflammatory role. Conversely, in the spleen of ASF-mice, mifepristone increased pro-inflammatory cytokines (TNF-α and IL-1ß), demonstrating an anti-inflammatory role. Furthermore, irrespective of sleep fragmentation, mifepristone increased pro-inflammatory cytokine gene expression in heart (IL-1ß), pre-frontal cortex (IL-1ß), and hypothalamus (IL-1ß). The results provide mixed evidence for pro- and anti-inflammatory functions of corticosterone to regulate inflammatory responses to acute sleep loss.
Assuntos
Glucocorticoides , Metirapona , Camundongos Endogâmicos C57BL , Mifepristona , Privação do Sono , Animais , Masculino , Metirapona/farmacologia , Privação do Sono/metabolismo , Privação do Sono/tratamento farmacológico , Camundongos , Mifepristona/farmacologia , Glucocorticoides/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Corticosterona/sangue , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genéticaRESUMO
Social status directly affects the health of humans and other animals. Low status individuals receive more antagonistic encounters, have fewer supportive relationships and have worse health outcomes. However, the physiological and cellular processes that mediate the relationship between the social environment and health are incompletely known. Epigenetic regulation of the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine pathway that activates in response to stressors, may be one process that is sensitive to the social environment. Here, we experimentally manipulated plumage, a key social signal in female tree swallows (Tachycineta bicolor) and quantified methylation of four genes in the HPA axis before and after treatment. We found that dulling the white breast plumage affected methylation in one gene, CRHR1; however, the effect depended on the original brightness of the bird. Methylation in this gene was correlated with baseline corticosterone levels, suggesting that DNA methylation of CRHR1 helps regulate glucocorticoid production in this species. Methylation in two other genes, FKBP5 and GR, changed over the course of the experiment, independent of treatment. These results show that methylation of these genes is labile into adulthood and suggest that epigenetic regulation of the HPA axis could help birds respond to current environmental conditions.
Assuntos
Metilação de DNA , Plumas , Sistema Hipotálamo-Hipofisário , Receptores de Hormônio Liberador da Corticotropina , Andorinhas , Animais , Feminino , Plumas/fisiologia , Andorinhas/genética , Andorinhas/fisiologia , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/fisiologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Corticosterona/sangue , Corticosterona/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Sistema Hipófise-Suprarrenal/fisiologia , Epigênese Genética , Estresse Fisiológico/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismoRESUMO
Atopic dermatitis (AD), a prevalent chronic inflammatory disease with multifactorial etiology, features epidermal barrier defects and immune overactivation. Synthetic glucocorticoids (GCs) are widely prescribed for treating AD due to their anti-inflammatory actions; however, mechanisms are incompletely understood. Defective local GC signaling due to decreased production of endogenous ligand and/or GC receptor (GR) levels was reported in prevalent inflammatory skin disorders; whether this is a consequence or contributing factor to AD pathology is unclear. To identify the chromatin-bound cell-type-specific GR protein interactome in keratinocytes, we used rapid immunoprecipitation of endogenous proteins and mass spectrometry identifying 145 interactors that increased upon dexamethasone treatment. GR-interacting proteins were enriched in p53/p63 signaling, including epidermal transcription factors with critical roles in AD pathology. Previous analyses indicating mirrored AD-like phenotypes between P63 overexpression and GR loss in epidermis, and our data show an intricate relationship between these transcription factors in human keratinocytes, identifying TP63 as a direct GR target. Dexamethasone treatment counteracted transcriptional up-regulation of inflammatory markers by IL4/IL13, known to mimic AD, causing opposite shifts in GR and P63 genomic binding. Indeed, IL4/IL13 decreased GR and increased P63 levels in cultured keratinocytes and human epidermal equivalents (HEE), consistent with GR down-regulation and increased P63 expression in AD lesions vs normal skin. Moreover, GR knockdown (GRKD) resulted in constitutive increases in P63, phospho-P38 and S100A9, IL6, and IL33. Also, GRKD culture supernatants showed increased autocrine production of TH2-/TH1-/TH17-TH22-associated factors including IL4, CXCL10, CXCL11, and CXCL8. GRKD HEEs showed AD-like features including hyperplasia and abnormal differentiation, resembling phenotypes observed with GR antagonist or IL4/IL13 treatment. The simultaneous GR/P63 knockdown partially reversed constitutive up-regulation of inflammatory genes in GRKD. In summary, our data support a causative role for GR loss in AD pathogenesis via functional interactions with P63 and autocrine signaling in epidermal keratinocytes.