RESUMO
Standigm ASK™ revolutionizes healthcare by addressing the critical challenge of identifying pivotal target genes in disease mechanisms-a fundamental aspect of drug development success. Standigm ASK™ integrates a unique combination of a heterogeneous knowledge graph (KG) database and an attention-based neural network model, providing interpretable subgraph evidence. Empowering users through an interactive interface, Standigm ASK™ facilitates the exploration of predicted results. Applying Standigm ASK™ to idiopathic pulmonary fibrosis (IPF), a complex lung disease, we focused on genes (AMFR, MDFIC and NR5A2) identified through KG evidence. In vitro experiments demonstrated their relevance, as TGFß treatment induced gene expression changes associated with epithelial-mesenchymal transition characteristics. Gene knockdown reversed these changes, identifying AMFR, MDFIC and NR5A2 as potential therapeutic targets for IPF. In summary, Standigm ASK™ emerges as an innovative KG and artificial intelligence platform driving insights in drug target discovery, exemplified by the identification and validation of therapeutic targets for IPF.
Assuntos
Inteligência Artificial , Fibrose Pulmonar Idiopática , Humanos , Reconhecimento Automatizado de Padrão , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Pulmão/metabolismoRESUMO
Numerous efforts in basic and clinical studies have explored the potential anti-aging and health-promoting effects of NAD+-boosting compounds such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). Despite these extensive efforts, our understanding and characterization of their whole-body pharmacodynamics, impact on NAD+ tissue distribution, and mechanism of action in various tissues remain incomplete. In this study, we administered NMN via intraperitoneal injection or oral gavage and conducted a rigorous evaluation of NMN's pharmacodynamic effects on whole-body NAD+ homeostasis in mice. To provide more confident insights into NMN metabolism and NAD+ biosynthesis across different tissues and organs, we employed a novel approach using triple-isotopically labeled [18O-phosphoryl-18O-carbonyl-13C-1-ribosyl] NMN. Our results provide a more comprehensive characterization of the NMN impact on NAD+ concentrations and absolute amounts in various tissues and the whole body. We also demonstrate that mice primarily rely on the nicotinamide and NR salvage pathways to generate NAD+ from NMN, while the uptake of intact NMN plays a minimal role. Overall, the tissue-specific pharmacodynamic effects of NMN administration through different routes offer novel insights into whole-body NAD+ homeostasis, laying a crucial foundation for the development of NMN as a therapeutic supplement in humans.
Assuntos
NAD , Mononucleotídeo de Nicotinamida , Humanos , Camundongos , Animais , Mononucleotídeo de Nicotinamida/farmacologia , Mononucleotídeo de Nicotinamida/metabolismo , NAD/metabolismo , Envelhecimento , Transporte BiológicoRESUMO
Cushing's syndrome (CS) caused by hypercortisolism is occasionally accompanied by metabolic disorders such as hypertension, diabetes mellitus (DM), dyslipidemia, and central obesity. Thus morbidity and mortality, observed in cardiovascular disease, are elevated in patients with CS. We hypothesized that HDAC inhibition (HDACi) decreased transcriptional activity of glucocorticoid receptor (GR), which ameliorates hypertension and hyperglycemia in patients with CS. To establish an animal model of hypercortisolism, Sprague-Dawley rats were infused with adrenocorticotropic hormone (ACTH, 40 ng/day) or dexamethasone (Dex, 10 µg/day) via osmotic minipumps for 4 wk. Expression of GR target genes was determined by quantitative real-time PCR (qRT-PCR). GR enrichment on specific loci, and across the whole genome, was analyzed by chromatin immunoprecipitation (ChIP) and ChIPseq, respectively. HDACi decreased blood pressure and expression of ion regulators in the kidneys of ACTH-infused rats. Additionally, HDACi reduced deposition of polysaccharide, fasting blood glucose level, glucose intolerance, and expression of gluconeogenesis genes in the livers and kidneys of ACTH- and Dex-infused rats. Among class I HDACs, HDAC1 and HDAC3 interacted with GR. HDAC1 knockdown resulted in increased level of acetylation and decreased transcriptional activity of GR. GR recruitment on the promoters of 2,754 genes, which include ion transporters, channels, and gluconeogenic genes, was significantly decreased by MS-275, a class I HDAC inhibitor. These results indicate that HDACi ameliorates hypertension and hyperglycemia in a model of CS by decreasing the transcriptional activity of GR via elevating its level of acetylation.
Assuntos
Glicemia/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Síndrome de Cushing/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Hiperglicemia/prevenção & controle , Hipertensão/prevenção & controle , Animais , Glicemia/metabolismo , Síndrome de Cushing/sangue , Síndrome de Cushing/patologia , Síndrome de Cushing/fisiopatologia , Modelos Animais de Doenças , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hiperglicemia/sangue , Hiperglicemia/complicações , Hiperglicemia/fisiopatologia , Hipertensão/sangue , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Cushing's syndrome (CS) is a collection of symptoms caused by prolonged exposure to excess cortisol. Chronically elevated glucocorticoid (GC) levels contribute to hepatic steatosis. We hypothesized that histone deacetylase inhibitors (HDACi) could attenuate hepatic steatosis through glucocorticoid receptor (GR) acetylation in experimental CS. To induce CS, we administered adrenocorticotropic hormone (ACTH; 40 ng/kg/day) to Sprague-Dawley rats by subcutaneous infusion with osmotic mini-pumps. We administered the HDACi, sodium valproate (VPA; 0.71% w/v), in the drinking water. Treatment with the HDACi decreased steatosis and the expression of lipogenic genes in the livers of CS rats. The enrichment of GR at the promoters of the lipogenic genes, such as acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn), and sterol regulatory element binding protein 1c (Srebp1c), was markedly decreased by VPA. Pan-HDACi and an HDAC class I-specific inhibitor, but not an HDAC class II a-specific inhibitor, attenuated dexamethasone (DEX)-induced lipogenesis in HepG2 cells. The transcriptional activity of Fasn was decreased by pretreatment with VPA. In addition, pretreatment with VPA decreased DEX-induced binding of GR to the glucocorticoid response element (GRE). Treatment with VPA increased the acetylation of GR in ACTH-infused rats and DEX-induced HepG2 cells. Taken together, these results indicate that HDAC inhibition attenuates hepatic steatosis hrough GR acetylation in experimental CS.
RESUMO
Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1), and atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic minipumps containing ACTH (40 ng·kg-1·day-1). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, atrogin-1, and FOXO3a were upregulated and the FOXO3a promoter was targeted by the glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a were significantly decreased by the GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on the promoters. Knockdown of FOXO3a prevented dexamethasone-induced MuRF-1 and atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and atrogin-1 in Cushing's syndrome.
Assuntos
Síndrome de Cushing/metabolismo , Modelos Animais de Doenças , Proteína Forkhead Box O3/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/etiologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Síndrome de Cushing/patologia , Síndrome de Cushing/fisiopatologia , Proteína Forkhead Box O3/agonistas , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/agonistas , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Interferência de RNA , Ratos Sprague-Dawley , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta/efeitos dos fármacos , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Proteínas Ligases SKP Culina F-Box/genética , Proteínas com Motivo Tripartido/agonistas , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genéticaRESUMO
A mutation in the mineralocorticoid receptor (MRS 810L ) leads to early-onset hypertension, which is markedly exacerbated during pregnancy. The mutation causes progesterone and even the MR antagonist spironolactone to become potent agonists. Thus, it is hard to control hypertension in patients harbouring this mutation. We hypothesized that histone deacetylase inhibition (HDACi), but not the MR antagonist spironolactone, attenuates atypical transcriptional activity of activating mutant MR (MRS 810L ). We established HEK293T cells overexpressing wild-type MR (MRWT ) or MRS 810L and determined their transcriptional activities by luciferase assay. Expression of MR target genes was measured by quantitative real-time PCR (qRT-PCR). Treatment with aldosterone increased the expression of MR target genes as well as the transcriptional activities in HEK293T cells transfected either with MRWT or MRS 810L . Treatment with either spironolactone or progesterone also increased the expression of MR target genes as well as transcriptional activity, but only in HEK293T cells transfected with MRS 810L . Spironolactone abolished the promoter activity stimulated by aldosterone in HEK293T cells transfected with MRWT . Treatment with HDAC inhibitors attenuated the transcriptional activity as well as the expression of MR target genes induced by aldosterone, spironolactone, or progesterone whether HEK293T cells were transfected with either MRWT or MRS 810L . These results indicate that HDACi, but not an MR antagonist spironolactone, attenuates atypical transcriptional activity of an activating mutant MR (MRS 810L ).
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Mutação/fisiologia , Receptores de Mineralocorticoides/fisiologia , Espironolactona/farmacologia , Transcrição Gênica/fisiologia , Aldosterona/farmacologia , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Progesterona/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.
RESUMO
Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.
Assuntos
Acetilação/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Fibrose/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Receptores de Mineralocorticoides/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Imunoprecipitação da Cromatina/métodos , Fibrose/genética , Fibrose/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Masculino , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Mineralocorticoides/genéticaRESUMO
OBJECTIVE: This study evaluated the influence of surface characteristics of various denture lining materials on the adherence of Candida albicans. MATERIALS AND METHODS: Four different types of materials (tissue conditioners, acrylic and silicone soft liners and hard reline materials) were selected. Disk-shaped material specimens were prepared and their surface roughness values (R(a) ) measured using a profilometer. The contact angles of four reference liquids were measured on the material surfaces and surface energy parameters (total surface energy, acid and base components, degree of hydrophobicity/hydrophilicity) of the materials were calculated in accordance with acid-base theory. Specimens were incubated with C. albicans and adhering fungi quantified using the colony counting method. Data were statistically analyzed using a one-way ANOVA with Games-Howell post-hoc test (α = 0.05). Pearson correlation analysis was applied to detect correlations between surface characteristics and Candida adhesion. RESULTS: Significant differences in the surface roughness of the materials were found (p < 0.001). The acrylic soft liners were more hydrophilic than the other materials. Overall, the acrylic soft liners and tissue conditioners showed significantly greater Candida adhesion than silicone soft liners and hard reline materials (p < 0.05). The Pearson correlation analysis indicated that the base component and degree of hydrophobicity/hydrophilicity of the materials (p = 0.005/0.008), rather than the total surface energy and the surface roughness (p = 0.093/0.057), affected C. albicans adherence in a statistically significant way. CONCLUSIONS: The adhesion of C. albicans to denture lining materials can be accounted for in terms of interfacial acid-base interactions.
Assuntos
Candida albicans/fisiologia , Adesão Celular , Materiais Dentários , Reembasadores de Dentadura/microbiologia , Equilíbrio Ácido-Base , Resinas Acrílicas , Análise de Variância , Contagem de Colônia Microbiana , Interações Hidrofóbicas e Hidrofílicas , Elastômeros de Silicone , Estatísticas não Paramétricas , Propriedades de Superfície , Condicionamento de Tecido Mole OralRESUMO
Combined oral contraceptive (COC) use is associated with increased risk of developing hypertension. Activation of the intrarenal renin-angiotensin system (RAS) and endothelial dysfunction play an important role in the development of hypertension. We tested the hypothesis that COC causes hypertension that is associated with endothelial dysfunction and upregulation of intrarenal angiotensin-converting enzyme 1 (Ace1) and angiotensin II type 1 receptor (At1r). Female Sprague-Dawley rats aged 12 weeks received (p.o.) olive oil (control) and a combination of 0.1 µg ethinylestradiol and 1.0 µg norgestrel (low COC) or 1.0 µg ethinylestradiol and 10.0 µg norgestrel (high COC) daily for 6 weeks. Blood pressure was recorded by tail cuff plethysmography. Expression of genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. COC treatment led to increased blood pressure, circulating uric acid, C-reactive protein and plasminogen activator inhibitor-1, renal uric acid, and expression of renal Ace1 and At1r. COC treatment resulted in increased contractile responses to phenylephrine in endothelium-denuded aortic rings. Endothelium-dependent relaxation responses to acetylcholine, but not endothelium-independent relaxation responses to nitric oxide (NO) donation by sodium nitroprusside, were attenuated in COC-exposed rings. Impaired relaxation responses to acetylcholine were masked by the presence of NO synthase inhibitor (L-NAME) in the COC-exposed rings, whereas the responses to acetylcholine in the presence of selective cyclooxygenase-2 inhibitor (NS-398) were enhanced. These findings indicate that COC induces hypertension that is accompanied by endothelial dysfunction, upregulated intrarenal Ace1 and At1r expression, and elevated proinflammatory biomarkers.
Assuntos
Endotélio Vascular/fisiopatologia , Combinação Etinil Estradiol e Norgestrel , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Córtex Renal/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Vasoconstrição , Vasodilatação , Animais , Pressão Sanguínea , Anticoncepcionais Orais Combinados , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Epoprostenol/metabolismo , Feminino , Hipertensão/induzido quimicamente , Hipertensão/genética , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismo , Peptidil Dipeptidase A/metabolismo , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Regulação para Cima , Ácido Úrico/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Clinical studies have shown that the use of combined oral contraceptive in pre-menopausal women is associated with fluid retention. However, the molecular mechanism is still elusive. We hypothesized that combined oral contraceptive (COC) ethinyl estradiol (EE) and norgestrel (N) synergistically activates mineralocorticoid receptor (MR) through histone code modifications. Twelve-week-old female Sprague-Dawley rats were treated with olive oil (control), a combination of 0.1µg EE and 1.0µg N (low COC) or 1.0µg EE and 10.0µg N (high COC) as well as 0.1 or 1.0µg EE and 1.0 or 10.0µg N daily for 6 weeks. Expression of MR target genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. MR was quantified by western blot. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes as well as histone code modifications was analyzed by chromatin immunoprecipitation assay. Treatment with COC increased renal cortical expression of MR target genes such as serum and glucocorticoid-regulated kinase 1 (Sgk-1), glucocorticoid-induced leucine zipper (Gilz), epithelial Na(+)channel (Enac) and Na(+)-K(+)-ATPase subunit α1 (Atp1a1). Although COC increased neither serum aldosterone nor MR expression in kidney cortex, it increased recruitment of MR and Pol II in parallel with increased H3Ac and H3K4me3 on the promoter regions of MR target genes. However, treatment with EE or N alone did not affect renal cortical expression of Sgk-1, Gilz, Enac or Atp1a1. These results indicate that COC synergistically activates MR through histone code modifications.
Assuntos
Anticoncepcionais Orais Combinados/farmacologia , Código das Histonas/efeitos dos fármacos , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/metabolismo , Animais , Sequência de Bases , Sinergismo Farmacológico , Etinilestradiol/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Norgestrel/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/efeitos dos fármacos , RNA Polimerase II/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genéticaRESUMO
Histone deacetylases (HDACs) act as corepressors in gene transcription by altering the acetylation of histones, resulting in epigenetic gene silencing. We previously reported that HDAC3 acts as a coactivator of the mineralocorticoid receptor (MR). Although HDAC3 forms complexes with class II HDACs, their potential role in the transcriptional activity of MR is unclear. We hypothesized that HDAC4 of the class II family stimulates the transcriptional activity of MR. The expression of MR target genes was measured by quantitative real-time PCR. MR and RNA polymerase II recruitment to promoters of MR target genes was analyzed by chromatin immunoprecipitation. The association of MR with HDACs was investigated by co-immunoprecipitation. MR acetylation was determined with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. Among the class II HDACs, HDAC4 interacted with both MR and HDAC3 after aldosterone stimulation. The nuclear translocation of HDAC4 was mediated by protein kinase A (PKA) and protein phosphatases (PP). The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568). MR acetylation was increased by H89, calyculin A, and MS-275, but not by MC1568. Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA. A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3. Thus, we have uncovered a crucial role for a class II HDAC in the activation of MR-dependent transcription.