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BACKGROUND: This study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal-Wallis and Dunn. RESULTS: Echocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *# nmol/g tissue; * p < 0.05 vs S, # p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66-1.66); T 0.78 (0.61-1.04) *; SNAC 1.07 (1.01-1.38); TNAC 1.06 (1.01-1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups. CONCLUSION: N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.
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Acetilcisteína , Hipertensão , NADPH Oxidases , Estresse Oxidativo , Ratos Endogâmicos SHR , Remodelação Ventricular , Animais , Masculino , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Hipertensão/fisiopatologia , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , Ratos , Antioxidantes/farmacologia , Condicionamento Físico Animal , Modelos Animais de Doenças , NADPH Oxidase 2/metabolismo , NADPH Oxidase 2/genética , NADPH Oxidase 4/metabolismo , NADPH Oxidase 4/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Miocárdio/metabolismo , Miocárdio/patologia , Peróxidos Lipídicos/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Suplementos Nutricionais , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Hipertrofia Ventricular Esquerda/metabolismoRESUMO
BACKGROUND/AIMS: To evaluate the effects of the antioxidant N-acetylcysteine (NAC) on cardiac structure and function in rats with long-term ascending aortic stenosis (AS). METHODS: Four months after inducing AS, Wistar rats were assigned into the groups Sham, AS, and AS treated with NAC (AS-NAC) and followed for eight weeks. Cardiac structure and function were evaluated by echocardiogram. Myocardial antioxidant enzymes activity was measured by spectrophotometry and malondialdehyde serum concentration by HPLC. Gene expression of NADPH oxidase subunits NOX2, NOX4, p22 phox, and p47 phox was assessed by real time RT-PCR and protein expression of MAPK proteins by Western blot. Statistical analyzes were performed with Goodman and ANOVA or Mann-Whitney Results: NAC restored myocardial total glutathione (Sham 20.8±3.00; AS 12.6±2.92; AS-NAC 17.6±2.45 nmol/g tissue; p<0.05 AS vs Sham and AS-NAC). Malondialdehyde serum concentration was lower in AS-NAC and myocardial lipid hydroperoxide was higher in AS (Sham 199±48.1; AS 301±36.0; AS-NAC 181±41.3 nmol/g tissue). Glutathione peroxidase activity was lower in AS than Sham. Echocardiogram showed LV concentric hypertrophy with systolic and diastolic dysfunction before and after treatment; no differences were observed between AS-NAC and AS groups. NAC reduced p-ERK and p-JNK protein expression, attenuated myocardial fibrosis, and decreased the frequency of right ventricular hypertrophy. CONCLUSION: N-acetylcysteine restores myocardial total glutathione, reduces systemic and myocardial oxidative stress, improves MAPK signaling, and attenuates myocardial fibrosis in aortic stenosis rats.
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Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Estenose da Valva Aórtica/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Animais , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Glutationa/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Ratos WistarRESUMO
INTRODUCTION: Exercise is an important therapeutic strategy for preventing and treating myocardial infarction (MI)-induced cardiac remodeling and heart failure. However, the myocardial effects of resistance exercise on infarcted hearts are not completely established. In this study, we investigated the effects of resistance exercise on structural, functional, and molecular cardiac alterations in infarcted rats. METHODS: Three months after MI induction or simulated surgery, Wistar rats were assigned into three groups: Sham (n = 14); MI (n = 9); and exercised MI (MI-Ex, n = 13). Exercised rats performed, 3 times a week for 12 weeks, four climbs on a ladder with progressive loads. Cardiac structure and left ventricle (LV) function were analyzed by echocardiogram. Myocyte diameters were evaluated in hematoxylin- and eosin-stained histological sections as the smallest distance between borders drawn across the nucleus. Myocardial energy metabolism, lipid hydroperoxide, malondialdehyde, protein carbonylation, and antioxidant enzyme activities were evaluated by spectrophotometry. Gene expressions of NADPH oxidase subunits were evaluated by RT-PCR. Statistical analyses were performed using ANOVA and Tukey or Kruskal-Wallis and Dunn's test. RESULTS: Mortality did not differ between the MI-Ex and MI groups. MI had dilated left atrium and LV, with LV systolic dysfunction. Exercise increased the maximum load-carrying capacity, with no changes in cardiac structure or LV function. Myocyte diameters were lower in MI than in Sham and MI-Ex. Lactate dehydrogenase and creatine kinase activity were lower in MI than in Sham. Citrate synthase and catalase activity were lower in MI and MI-Ex than in Sham. Lipid hydroperoxide concentration was lower in MI-Ex than in MI. Nox2 and p22phox gene expressions were higher in MI-Ex than in Sham. Gene expression of Nox4 was higher in MI and MI-Ex than in Sham, and p47phox was lower in MI than in Sham. CONCLUSION: Late resistance exercise was safe in infarcted rats. Resistance exercise improved maximum load-carrying capacity, reduced myocardial oxidative stress, and preserved myocardial metabolism, with no changes in cardiac structure or left ventricle function in infarcted rats.
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OBJECTIVES: Preeclampsia (PE) is an important syndrome of gestation characterized by placental and systemic inflammation. High plasma concentration of uric acid are frequently associated with inflammation and endothelial dysfunction and may contribute to PE pathogenesis. This study aimed to evaluate the vitamin D (VD) immunomodulatory effect on the NLRP1/NLRP3 inflammasomes in placental explants from preeclamptic (PE) and normotensive (NT) pregnant women. STUDY DESIGN: Placental explants from 10 late-onset PE (LOPE), 10 early-onset PE (EOPE), and 10 NT pregnant women were cultured with or without monosodium urate (MSU) and VD. MAIN OUTCOME MEASURES: Gene and protein expression of NLRP1, NLRP3, HMGB1, caspase-1, interleukin-1 beta (IL-1ß), and IL-18 were determined by quantitative PCR and Western blotting/ELISA. Statistical significance was accepted at p < 0.05. RESULTS: Basal gene and protein expression of NLRP1/NLRP3 and IL-1ß, IL-18 and HMGB1 were significantly higher in explants from EOPE compared to LOPE and NT pregnant women. In addition, culture with MSU increased these inflammatory markers, and concomitant treatment with MSU+VD decreased this effect. CONCLUSIONS: The results demonstrated that NLRP1 and NLRP3 inflammasomes are upregulated in the placental tissue of EOPE women, associated with high production of inflammatory cytokines. The in vitro treatment with VD downregulated placental inflammasomes induced by MSU, suggesting its immunomodulatory role in the systemic inflammation of PE.
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Proteína HMGB1 , Pré-Eclâmpsia , Feminino , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação , Interleucina-18 , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placenta/metabolismo , Gravidez , Ácido Úrico/farmacologia , Vitamina D , VitaminasRESUMO
Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.
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The placenta is a temporary organ that plays critical roles at the maternal-fetal interface. Normal development and function of the placenta is dependent on hormonal signaling pathways that make the placenta a target of endocrine disrupting chemical (EDC) action. Studies showing association between prenatal exposure, hormone disruption, and reproductive damage indicate that EDCs are developmentally toxic and can impact future generations. In this context, new placental models (trophoblast-derived cell lines, organotypic or 3D cell models, and physiologically based kinetic models) have been developed in order to create new approach methodology (NAM) to assess and even prevent such disastrous toxic harm in future generations. With the widespread discouragement of conducting animal studies, it has become irrefutable to develop in vitro models that can serve as a substitute for in vivo models. The goal of this review is to discuss the newest in vitro models to understand the maternal-fetal interface and predict placental development, physiology, and dysfunction generated by failures in molecular hormone control mechanisms, which, consequently, may change epigenetic programming to increase susceptibility to metabolic and other disorders in the offspring. We summarize the latest placental models for developmental toxicology studies, focusing mainly on three-dimensional (3D) culture models.
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Desenvolvimento Fetal , Placenta , Animais , Feminino , Desenvolvimento Fetal/fisiologia , Hormônios/metabolismo , Placenta/metabolismo , Placentação , Gravidez , TrofoblastosRESUMO
Adiponectin and leptin, important for metabolic regulation, are synthesized and secreted by adipose tissue and are influenced by triiodothyronine (T3) that activates the MAPK/ERK and integrin αVß3 pathways, modulating gene expression. Adipocytes were treated with T3 (10 nM), for 1 h, in the absence or presence of PD98059 (PD) and tetraiodothyroacetic acid (Tetrac), which are pathways inhibitors. The cells were incubated with Adipo Red/Oil Red O reagents, and intracellular lipid accumulation [glycerol and triacylglycerol (TAG)], MTT, 8-hydroxideoxyguanosine (8-OH-dG), and mRNA and protein expression were assessed. T3 increased leptin mRNA and protein expression, and, in contrast, there was a decrease in the Tetrac + T3 group. Adiponectin mRNA expression was not altered by T3, though it had increased its protein expression, which was terminated by inhibitors PD + T3 and Tetrac + T3. However, T3 did not alter PPARγ protein expression, lipid accumulation, TAG, glycerol, and DNA damage, but PD + T3 and Tetrac + T3 reduced these parameters. T3 activated the MAPK/ERK pathway on adipocytes to modulate the adiponectin protein expression and integrin αvß3 to alter the leptin gene expression.
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Adipócitos/efeitos dos fármacos , Adiponectina/metabolismo , Leptina/metabolismo , Tri-Iodotironina/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Expressão Gênica/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Tiroxina/análogos & derivados , Tiroxina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Triiodothyronine (T3) and irisin (I) can modulate metabolic status, increase heat production, and promote differentiation of white adipose tissue (WAT) into brown adipose tissue (BAT). Herein, human subcutaneous white adipocytes were treated with 10 nM T3 or 20 nM I for 24 h to evaluate intracellular lipid accumulation, triglyceride, and glycerol levels, oxidative stress, DNA damage, and protein levels of uncoupling protein 1 (UCP1), adiponectin, leptin, peroxisome proliferator-activated receptor gamma (PPARγ), and fibronectin type III domain-containing protein 5 (FNDC5). T3 and irisin improved UCP1 production, lipid profile, oxidative stress, and DNA damage. T3 elevated adiponectin and leptin levels with a concomitant decrease in PPARy and FNDC5 levels. However, irisin did not alter adipokine, PPARy, and FNDC5 levels. The results indicate that T3 may be used to increase leptin and adiponectin levels to improve insulin sensitivity, and irisin may be used to prevent obesity or maintain weight due to its impact on the lipid profile without altering adipokine levels.
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Adipócitos Brancos/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/fisiologia , Adipócitos Brancos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Transdiferenciação Celular/genética , Células Cultivadas , Fibronectinas/fisiologia , Expressão Gênica/efeitos dos fármacos , Glicerol/metabolismo , Humanos , Leptina/genética , Leptina/metabolismo , Metabolismo dos Lipídeos/genética , Lipólise/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Gordura Subcutânea/citologia , Gordura Subcutânea/efeitos dos fármacos , Gordura Subcutânea/fisiologia , Triglicerídeos/metabolismo , Tri-Iodotironina/fisiologia , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.
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Armadilhas Extracelulares , Paracoccidioides , Paracoccidioidomicose , Desoxirribonucleases , Humanos , Neutrófilos , Paracoccidioides/genéticaRESUMO
BACKGROUND: Corticosteroid injections in or around tendons for the treatment of athletic injuries are a common practice among orthopaedic surgeons and are apparently efficacious in the short term, although controversies persist related to local complications. PURPOSE: This study evaluated short-term (48 hours) biomechanical, biochemical, and histological alterations after a single injection of betamethasone into the normal tendons of rabbits. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 72 New Zealand White rabbits were randomly divided into 2 groups: the test group-in which 36 animals underwent 1 intratendinous injection of betamethasone (1.4 mg / 0.2 mL) in the right calcaneal tendon; the control group-in which the right calcaneal tendon of 36 animals was injected with saline (placebo control group) and the left calcaneal tendon was left untreated for normal standards (normal control). Forty-eight hours later, animals were euthanized and tendons were harvested. Metalloproteinase (MMP1 and MMP2) and interleukin (IL1 and IL6) expression levels, biomechanical resistance (load × elongation parameters), and histomorphometry (hematoxylin and eosin and picrosirius red stains for collagen fibers, tenocytes, and inflammatory cells) were analyzed in the tendons. RESULTS: The test group had a significant reduction in MMP2 expression as compared with the control groups ( P = .027). Regarding the other parameters, there were no additional significant differences between the groups. CONCLUSION: A single injection of corticosteroid into normal calcaneal tendons did not trigger acute local morphological, structural, or biomechanical injuries at 48 hours, but it did promote a significant decrease in MMP2 levels. Additional studies are needed with increased duration of follow-up, various doses, and multiple injections and in tendinopathic models. CLINICAL RELEVANCE: Some previous studies demonstrated early structural changes in tendons after a single corticosteroid injection, which was not corroborated by the present study. Metalloproteinase decrease is usually associated with a reduction in collagen degradation, which would be protective for the healing process. More studies are necessary to confirm the possible beneficial effect of these results in the long term and for tendinopathies.
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Tendão do Calcâneo/efeitos dos fármacos , Betametasona/administração & dosagem , Glucocorticoides/administração & dosagem , Traumatismos dos Tendões/tratamento farmacológico , Tendão do Calcâneo/enzimologia , Tendão do Calcâneo/lesões , Corticosteroides , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Injeções , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Coelhos , Distribuição Aleatória , Tendinopatia , Traumatismos dos Tendões/metabolismo , CicatrizaçãoRESUMO
OBJECTIVE: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway. MATERIALS AND METHODS: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. RESULTS: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. CONCLUSION: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.
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Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Fator de Crescimento Transformador alfa/genética , Tri-Iodotironina/genética , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/metabolismo , Feminino , Humanos , Células MCF-7/metabolismo , Proto-Oncogene Mas , Proto-Oncogenes/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
Triiodothyronine (T3) and estrogen (E2) play important roles in the bone remodeling process and signaling of receptor activator of the nuclear factor-kappa ß (RANKL) and osteoprotegerin (OPG) expressed by osteoblasts. However, little is known of the molecular action of these hormones in conditions of hyperthyroidism and associated E2 in human cells. AIMS: This study evaluated the effects of the physiological concentration of E2 (10â¯nM), alone or in association with physiological (1â¯nM) and supraphysiological (10â¯nM) concentrations of T3, on RANKL and OPG gene expression in human osteoblasts. MAIN METHODS: Alkaline phosphatase and osteocalcin assays were performed to verify the presence of mature osteoblasts. After mimicking the experimental hyperthyroidism in osteoblasts untreated or treated with E2, RANKL and OPG gene expression was analyzed by real-time PCR and protein expression by western Blot and ELISA. Alizarin Red staining analyzed the amount of bone matrix after hormonal treatments. KEY FINDINGS: E2 enhanced the gene expression of OPG when associated with 1â¯nM and 10â¯nMâ¯T3. E2 was able to restore the bone matrix after an initial decrease using 1â¯nM and 10â¯nMâ¯T3. The protective effect of E2 on the RANKL and OPG signaling pathway was demonstrated. E2 restored the bone matrix induced by experimental hyperthyroidism. SIGNIFICANCE: The data highlight the importance of E2 to maintain OPG expression and osteoblast activity against possible loss of bone mass, especially in conditions where T3 is in excess.
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Remodelação Óssea/efeitos dos fármacos , Estrogênios/fisiologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Remodelação Óssea/fisiologia , Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Estrogênios/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertireoidismo/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/fisiologiaRESUMO
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
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Tecido Adiposo/citologia , Sobrevivência Celular , Osteoblastos/citologia , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Colágeno Tipo III/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência , Modelos Biológicos , Osteoblastos/metabolismo , Osteopontina/metabolismo , Ligante RANK/metabolismo , Vimentina/metabolismoRESUMO
ABSTRACT Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway Materials and methods: The cell line was treated with T3 at a physiological dose (10−9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.