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1.
Nucleic Acids Res ; 48(18): 10342-10352, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32894284

RESUMO

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.


Assuntos
DNA Ribossômico/genética , Proteínas Hedgehog/genética , RNA Polimerase I/genética , Neoplasias de Mama Triplo Negativas/radioterapia , Proteína GLI1 em Dedos de Zinco/genética , Proteínas de Ciclo Celular/genética , Nucléolo Celular/genética , Nucléolo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , DNA Ribossômico/efeitos da radiação , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Humanos , RNA Polimerase I/efeitos da radiação , Radiação Ionizante , Ribossomos/genética , Ribossomos/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Transcrição Genética/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
2.
Mutat Res ; 856-857: 503220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32928367

RESUMO

We assessed the radioprotective and mitigative actions of sodium diclofenac, a non-steroidal anti-inflammatory drug using cultured human peripheral blood as a model. Both pre- and post-irradiation treatments with the drug reduced gamma radiation-induced formation of dicentric chromosome, cytochalasin-blocked micronuclei and γ-H2AX foci in human peripheral blood lymphocytes. This work supports the concept that sodium diclofenac may be a useful radiation countermeasure agent.


Assuntos
Diclofenaco/farmacologia , Relação Dose-Resposta à Radiação , Histonas/genética , Protetores contra Radiação/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Análise Citogenética/métodos , Reposicionamento de Medicamentos , Raios gama/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação
3.
Mutat Res ; 856-857: 503237, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32928370

RESUMO

Radiation has widespread applications in medicine. However, despite the benefits of medical radiation exposures, adverse long-term health effects are cause for concern. Protein and gene biomarkers are early indicators of cellular response after low-dose exposure. We examined DNA damage by quantifying γ-H2AX foci and expression of twelve candidate genes in the blood lymphocytes of patients exposed to low doses of X-radiation during neuro-interventional procedures. Entrance surface dose (ESD; 10.92-1062.55 mGy) was measured by thermoluminescence dosimetry (TLD). Absorbed dose was estimated using γ-H2AX focus frequency and gene expression, with in vitro dose-response curves generated for the same biomarkers. γ-H2AX foci in post-exposure samples were significantly higher than in pre-exposure samples. Among the genes analysed, FDXR, ATM, BCL2, MDM2, TNFSF9, and PCNA showed increased expression; CDKN1A, DDB2, SESN1, BAX, and TNFRSF10B showed unchanged or decreased expression. Absorbed dose, estimated based on γ-H2AX focus frequency and gene expression changes, did not show any correlation with measured ESD. Patients undergoing interventional procedures receive considerable radiation doses, resulting in DNA damage and altered gene expression. Medical procedures should be carried out using the lowest radiation doses possible without compromising treatment.


Assuntos
Histonas/efeitos da radiação , Linfócitos/efeitos da radiação , Imagem por Ressonância Magnética Intervencionista/efeitos adversos , Exposição à Radiação/efeitos adversos , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Histonas/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Pacientes , Doses de Radiação , Raios X/efeitos adversos
4.
Nucleic Acids Res ; 48(16): 9181-9194, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32789493

RESUMO

The NAD+-dependent deacetylase and mono-ADP-ribosyl transferase SIRT6 stabilizes the genome by promoting DNA double strand break repair, thereby acting as a tumor suppressor. However, whether SIRT6 regulates nucleotide excision repair (NER) remains unknown. Here, we showed that SIRT6 was recruited to sites of UV-induced DNA damage and stimulated the repair of UV-induced DNA damage. Mechanistic studies further indicated that SIRT6 interacted with DDB2, the major sensor initiating global genome NER (GG-NER), and that the interaction was enhanced upon UV irradiation. SIRT6 deacetylated DDB2 at two lysine residues, K35 and K77, upon UV stress and then promoted DDB2 ubiquitination and segregation from chromatin, thereby facilitating downstream signaling. In addition, we characterized several SIRT6 mutations derived from melanoma patients. These SIRT6 mutants ablated the stimulatory effect of SIRT6 on NER and destabilized the genome due to (i) partial loss of enzymatic activity (P27S or H50Y), (ii) a nonsense mutation (R150*) or (iii) high turnover rates (G134W). Overall, we demonstrate that SIRT6 promotes NER by deacetylating DDB2, thereby preventing the onset of melanomagenesis.


Assuntos
Carcinogênese/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Melanoma/genética , Sirtuínas/genética , Carcinogênese/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Melanoma/patologia , Mutação/efeitos da radiação , Ubiquitinação/efeitos da radiação , Raios Ultravioleta/efeitos adversos
5.
PLoS One ; 15(8): e0236689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785240

RESUMO

OBJECTIVE: To compare the effects of photobiomodulation therapy (PBMT) and pharmacological therapy (glucocorticoids and non-steroidal anti-inflammatory drugs) applied alone and in different combinations in mdx mice. METHODS: The animals were randomized and divided into seven experimental groups treated with placebo, PBMT, prednisone, non-steroidal anti-inflammatory drug (NSAIDs), PBMT plus prednisone and PBMT plus NSAID. Wild type animals were used as control. All treatments were performed during 14 consecutive weeks. Muscular morphology, protein expression of dystrophin and functional performance were assessed at the end of the last treatment. RESULTS: Both treatments with prednisone and PBMT applied alone or combined, were effective in preserving muscular morphology. In addition, the treatments with PBMT (p = 0.0005), PBMT plus prednisone (p = 0.0048) and PBMT plus NSAID (p = 0.0021) increased dystrophin gene expression compared to placebo-control group. However, in the functional performance the PBMT presented better results compared to glucocorticoids (p<0.0001). In contrast, the use of NSAIDs did not appear to add benefits to skeletal muscle tissue in mdx mice. CONCLUSION: We believe that the promising and optimistic results about the PBMT in skeletal muscle of mdx mice may in the future contribute to this therapy to be considered a safe alternative for patients with Duchenne Muscular Dystrophy (DMD) in a washout period (between treatment periods with glucocorticoids), allowing them to remain receiving effective and safe treatment in this period, avoiding at this way periods without administration of any treatment.


Assuntos
Distrofina/genética , Terapia com Luz de Baixa Intensidade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/efeitos da radiação , Distrofia Muscular de Duchenne/terapia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Terapia Combinada , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Glucocorticoides/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Prednisona/farmacologia
6.
PLoS One ; 15(6): e0234439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530967

RESUMO

Disturbance of circadian rhythms underlies various metabolic diseases. Constant light exposure (LL) is known to disrupt both central and peripheral circadian rhythms. Here, we attempted to determine whether the effects of LL are different between various peripheral tissues and whether time-restricted feeding restores the circadian rhythms especially in white adipose tissue (WAT). Six-week-old mice were subjected to three feeding regimes: ad libitum feeding under light/dark phase (LD), ad libitum feeding under LL cycle, and restricted feeding at night-time under LL cycle with a normal chow. After 3 weeks, we compared body weight, food intake, plasma levels of lipids and glucose, and the expression patterns of the clock genes and the genes involved in lipid metabolism in the liver and WAT. The mice kept under LL with or without time-restricted feeding were 5.2% heavier (p<0.001, n = 16) than the mice kept under LD even though the food intakes of the two groups were the same. Food intake occurred mostly in the dark phase. LL disrupted this pattern, causing disruptions in circadian rhythms of plasma levels of triglycerides (TG) and glucose. Time-restricted feeding partially restored the rhythms. LL eliminated the circadian rhythms of the expression of the clock genes as well as most of the genes involved in lipid metabolism in both liver and WAT. More notably, LL markedly decreased not only the amplitude but also the average levels of the expression of the genes in the liver, but not in the WAT, suggesting that transcription in the liver is sensitive to constant light exposure. Time-restricted feeding restored the circadian rhythms of most of the genes to various degrees in both liver and WAT. In conclusion, LL disrupted the peripheral circadian rhythms more severely in liver than in WAT. Time-restricted feeding restored the circadian rhythms in both tissues.


Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Jejum/fisiologia , Luz/efeitos adversos , Metabolismo dos Lipídeos/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Metabolismo dos Lipídeos/efeitos da radiação , Fígado/metabolismo , Masculino , Camundongos , Modelos Animais , Fotoperíodo
7.
Lasers Med Sci ; 35(8): 1841-1848, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32483748

RESUMO

Photobiomodulation (PBM) has been used to modulate the inflammatory and immune responses, pain relief, and to promote wound healing. PBM is widely used in dental practice and its cellular effects should be investigated. The aim was to evaluate if PBM changes proteins cell death-related, such as caspase-6 and Bcl-2, in periodontal ligament cells. Eighteen mice were divided in three groups (n = 6), i.e., (I) control, (II) 3 J cm-2, and (III) 30 J cm-2. Low power infrared laser (830 nm) parameters were power at 10 mW, energy densities at 3 and 30 J cm-2 in continuous emission mode, exposure time of 15 and 150 s, respectively for 4 days in a row. Twenty-four hours after last irradiation, the animals were euthanized, and their jaws were fixed and decalcified. Caspase-6 and Bcl-2 were analyzed by real-time polymerase chain reaction and immunocytochemical techniques, and DNA fragmentation was evaluated by TUNEL. Statistical differences were not significant to caspase-6 mRNA relative levels in tissues from jaws at both energy densities, but a significant increase of Bcl-2 mRNA relative levels was obtained at 30 J cm-2 group. Also, 30 J cm-2 group showed caspase-6 positive-labeled cells decreased and Bcl-2 positive-labeled cells significantly increased. TUNEL-labeled cells demonstrated DNA fragmentation decreased at 30 J cm-2. PBM can alter Bcl-2 mRNA relative level and both caspase-6 and Bcl-2 protein, modulating cell survival, as well as to reduce DNA fragmentation. More studies must be performed in order to obtain conclusive results about photobiostimulation effects using infrared low-level laser in apoptosis process as to achieve the optimum dosage.


Assuntos
Apoptose/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Ligamento Periodontal/citologia , Animais , Sobrevivência Celular/efeitos da radiação , Fragmentação do DNA/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , Cicatrização/efeitos da radiação
8.
Sci Rep ; 10(1): 7734, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382091

RESUMO

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico Sintase Tipo III/genética , Trombomodulina/genética , Fracionamento da Dose de Radiação , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Fatores de Transcrição MEF2/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/radioterapia , Radiação , Transdução de Sinais/efeitos da radiação
9.
Tissue Cell ; 63: 101329, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32223956

RESUMO

To investigate the protective function of low-level laser irradiation (LLLI) against ionizing irradiation and explore the molecular mechanism of photomodulation of Nrf2 protein, the impact of LLLI (635 nm, 5.7 J/cm2) before 2 Gy gamma ray radiation of radio-sensitive tissue hematopoietic stem cells was evaluated. As a result, reduced levels of reactive oxygen species and increased expression of antioxidant enzymes were detected. Moreover, increased expression of Nrf2 was observed after LLLI, whereas brusatol pretreatment before LLLI abolished this effect. In vivo, transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) was employed for therapy of hematopoietic function in an acute radiation sickness (H-ARS) mouse model, which was induced by 6-Gy ionizing irradiation; different hUC-MSC pretreatments including LLLI and Nrf2 RNAi were accounted for during experimental grouping. LLLI treatment of cells significantly increased the erythrocyte count and number of myelopoiesis clones (P < 0.05), but such improvements were reduced by Nrf2 RNAi pretreatment compared with cells transplanted without intervention. Therefore, LLLI may improve the radiation protection effect through molecular mechanisms related to the Nrf2 antioxidant pathway.


Assuntos
Proliferação de Células/efeitos da radiação , Células-Tronco Mesenquimais/efeitos da radiação , Fator 2 Relacionado a NF-E2/genética , Animais , Antioxidantes/metabolismo , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Lasers/efeitos adversos , Transplante de Células-Tronco Mesenquimais , Camundongos , Proteção Radiológica , Cordão Umbilical/efeitos da radiação
10.
Exp Hematol ; 84: 54-66, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240658

RESUMO

Exposure to high-dose total body irradiation (TBI) can result in hematopoietic acute radiation syndrome (H-ARS), characterized by leukopenia, anemia, and coagulopathy. Death from H-ARS occurs from hematopoietic insufficiency and opportunistic infections. Following radiation exposure, red blood cells (RBCs) undergo hemolysis from radiation-induced hemoglobin denaturation, causing the release of iron. Free iron can have multiple detrimental biological effects, including suppression of hematopoiesis. We investigated the impact of radiation-induced iron release on the bone marrow following TBI and the potential impact of the ACE inhibitor captopril, which improves survival from H-ARS. C57BL/6J mice were exposed to 7.9 Gy, 60Co irradiation, 0.6 Gy/min (LD70-90/30). RBCs and reticulocytes were significantly reduced within 7 days of TBI, with the RBC nadir at 14-21 days. Iron accumulation in the bone marrow correlated with the time course of RBC hemolysis, with an ∼10-fold increase in bone marrow iron at 14-21 days post-irradiation, primarily within the cytoplasm of macrophages. Iron accumulation in the bone marrow was associated with increased expression of genes for iron binding and transport proteins, including transferrin, transferrin receptor 1, ferroportin, and integrin αMß2. Expression of the gene encoding Nrf2, a transcription factor activated by oxidative stress, also increased at 21 days post-irradiation. Captopril did not alter iron accumulation in the bone marrow or expression of iron storage genes, but did suppress Nrf2 expression. Our study suggests that following TBI, iron is deposited in tissues not normally associated with iron storage, which may be a secondary mechanism of radiation-induced tissue injury.


Assuntos
Síndrome Aguda da Radiação/metabolismo , Medula Óssea/metabolismo , Raios gama/efeitos adversos , Hematopoese/efeitos da radiação , Ferro/metabolismo , Lesões Experimentais por Radiação/metabolismo , Síndrome Aguda da Radiação/genética , Síndrome Aguda da Radiação/patologia , Animais , Medula Óssea/patologia , Captopril/farmacologia , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/patologia
11.
Radiat Res ; 193(4): 351-358, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32126188

RESUMO

Millimeter waves (MMW) are broadband frequencies that have recently been used in several applications in wireless communications, medical devices and nonlethal weapons [i.e., the nonlethal weapon, Active Denial Systems, (ADS) operating at 94-95 GHz, CW]. However, little information is available on their potential effects on humans. These radio-frequencies are absorbed and stopped by the first layer of the skin. In this study, we evaluated the effects of 94 GHz on the gene expression of skin cells. Two rat populations consisting of 17 young animals and 14 adults were subjected to chronic long-term 94 GHz MMW exposure. Each group of animals was divided into exposed and sham subgroups. The two independent exposure experiments were conducted for 5 months with rats exposed 3 h per day for 3 days per week to an incident power density of 10 mW/cm2, which corresponded to twice the ICNIRP limit of occupational exposure for humans. At the end of the experiment, skin explants were collected and RNA was extracted. Then, the modifications to the whole gene expression profile were analyzed with a gene expression microarray. Without modification of the animal's temperature, long-term chronic 94 GHz-MMW exposure did not significantly modify the gene expression of the skin on either the young or adult rats.


Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Ondas de Rádio/efeitos adversos , Pele/efeitos da radiação , Tecnologia sem Fio , Animais , Humanos , Ratos , Ratos Pelados/genética , Ratos Pelados/metabolismo , Medição de Risco , Pele/metabolismo , Transcriptoma/efeitos da radiação
12.
J Nutr ; 150(5): 1100-1108, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133527

RESUMO

BACKGROUND: Radiotherapy inadvertently affects gastrointestinal (GI) epithelial cells, causing intestinal barrier disruption and increased permeability. OBJECTIVE: We examined the effect of amino acid-based oral rehydration solution (AA-ORS) on radiation-induced changes of intestinal barrier function and epithelial tight junctions (TJs) in a randomized experimental study using a total-body irradiation (TBI) mouse model. METHODS: Eight-week-old male Swiss mice received a single-dose TBI (0, 1, 3, or 5 Gy), and subsequent gastric gavage with AA-ORS (threonine, valine, serine, tyrosine, and aspartic acid) or saline for 2 or 6 d. Intestinal barrier function of mouse ileum was characterized by electrophysiological analysis of conductance, anion selectivity, and paracellular permeability [fluorescein isothiocyanate (FITC)-dextran]. Ultrastructural changes of TJs were evaluated by transmission electron microscopy. Membrane protein and mRNA expression of claudin-1, -2, -3, -5, and -7, occludin, and E-cadherin were analyzed with western blot, qPCR, and immunohistochemistry. Nonparametric tests were used to compare treatment-dose differences for each time point. RESULTS: Saline-treated mice had a higher conductance at doses as low as 3 Gy, and as early as 2 d post-TBI compared with 0 Gy (P < 0.001). Paracellular permeability and dilution potential were increased 6 d after 5 Gy TBI (P < 0.001). Conductance decreased with AA-ORS after 2 d in 3-Gy and 5-Gy mice (P < 0.05 and P < 0.001), and on day 6 after 5 Gy TBI (P < 0.001). Anion selectivity and FITC permeability decreased from 0.73 ± 0.02 to 0.61 ± 0.03 pCl/pNa (P < 0.01) and from 2.7 ± 0.1 × 105 to 2.1 ± 0.1 × 105 RFU (P < 0.001) in 5-Gy mice treated with AA-ORS for 6 d compared with saline. Irradiation-induced ultrastructural changes of TJs characterized by decreased electron density and gap formation improved with AA-ORS. Reduced claudin-1, -3, and -7 membrane expression after TBI recovered with AA-ORS within 6 d, whereas claudin-2 decreased indicating restitution of TJ proteins. CONCLUSIONS: Radiation-induced functional and structural disruption of the intestinal barrier in mice is reversed by AA-ORS rendering AA-ORS a potential treatment option in prospective clinical trials in patients with gastrointestinal barrier dysfunction.


Assuntos
Aminoácidos/administração & dosagem , Intestinos/efeitos da radiação , Soluções para Reidratação/química , Soluções para Reidratação/farmacologia , Junções Íntimas/efeitos da radiação , Animais , Hidratação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Masculino , Camundongos , Permeabilidade , RNA Mensageiro , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
13.
Lasers Med Sci ; 35(9): 1927-1936, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32162133

RESUMO

Phototherapy has been used to treat postoperative pain and inflammatory response in rheumatoid arthritis. Confidence in this approach, however, is impaired by lack of understanding of the light-triggered cellular and molecular mechanisms. The purpose of this study was to characterize the response of human synoviocyte MH7A cells to visible LED red light in an attempt to elucidate the associated action mechanism. Human synoviocyte MH7A cells were treated with 630-nm LED light after stimulation of tumor necrosis factor-α (TNF-α). The effects of light radiation on cell proliferation and migration were detected by MTT assay and scratch test. The expressions of inflammatory cytokines were measured using RT-qPCR. This was followed by detection of the levels of extracellular proteins IL-6 and IL-8 after differential radiation. Furthermore, the expression levels and activation of proteins on PI3K/AKT/mTOR signaling pathway were examined with Western blot. In terms of the proliferation and migration, repeated radiation with LED red light (630 nm, 26 and 39 J/cm2) exerted an inhibitory effect on synoviocyte MH7A cells. Expression of inflammatory factors (IL-6, IL-1ß, IL-8, and MMP-3) was reduced; meanwhile, the expression of anti-inflammatory factor IL-10 was promoted. At the protein level, treatment with 39 J/cm2 of LED red light could decrease the level of extracellular protein (IL-6 and IL-8) and affect the expression and phosphorylation of proteins on TRPV4/PI3K/AKT/mTOR signaling pathway induced by TNF-α. These results demonstrated that LED red light (630 nm) inhibits proliferation and migration of MH7A cells. The growth-inhibiting effects of LED red light on human synoviocyte MH7A cells appear to be associated with regulation of the TRPV4/PI3K/AKT/mTOR signaling pathway.


Assuntos
Terapia com Luz de Baixa Intensidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sinoviócitos/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPV/metabolismo , Linhagem Celular , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Citocinas/metabolismo , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Mediadores da Inflamação/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos da radiação , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismo , Sinoviócitos/patologia
14.
Int J Radiat Biol ; 96(6): 748-758, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32149567

RESUMO

Purpose: Simple, rapid and high-throughput dose assessment is critical for clinical diagnosis, treatment and emergency intervention in a large-scale radiological accident. The goal of this study is to screen and identify new ionizing radiation-responsive protein biomarkers in rat plasma.Materials and methods: Sprague-Dawley rats were exposed to single doses of 0, 1, 3, 5 Gy of Cobalt-60 γ-rays total body irradiation at a dose rate of 1 Gy/min. The tandem mass tag labeling (TMT) combined with liquid chromatography mass spectrometry (LC-MS/MS) approach was used to screen the differentially expressed proteins in rat plasma collected at 1, 3, 5 and 7 days post-irradiation. Bioinformatics analysis was conducted to explore the biological functions of these proteins. The expression levels of candidate radiation-sensitive protein biomarkers were confirmed using enzyme-linked immune-sorbent assay (ELISA).Results: A total of 503 differentially expressed proteins were identified. Most of these proteins were implicated in immune response, phagocytosis and signal transduction following ionizing radiation. Five up-regulated proteins including alpha-2-macroglobulin (A2m), chromogranin-A (CHGA), glutathione pertidase 3 (GPX3), clusterin (Clu) and ceruloplasmin (Cp) were selected for ELISA analysis. It was found that the expression levels of A2m, CHGA and GPX3 protein were increased in a dose-dependent manner at 1, 3 and 5 days after irradiation.Conclusion: Proteomics analysis revealed radiation-induced differentially expressed proteins in rat plasma. Our results suggested that A2m, CHGA, GPX3 protein expressions alterations in rat plasma may have potential as biomarkers to evaluate radiation exposure.


Assuntos
Proteínas Sanguíneas/metabolismo , Raios gama/efeitos adversos , Regulação da Expressão Gênica/efeitos da radiação , Animais , Biomarcadores/sangue , Proteínas Sanguíneas/genética , Ontologia Genética , Mapas de Interação de Proteínas/efeitos da radiação , Ratos , Ratos Sprague-Dawley
15.
DNA Cell Biol ; 39(5): 790-800, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32176536

RESUMO

Interleukin-1 beta (IL1B) is a key inducer of inflammation and an important factor in the regulation of hematopoietic stem cells and mesenchymal stromal progenitors. Irradiation of mice with ionizing radiation has been shown to induce a lasting increase in IL1B concentration in peripheral blood. One of the possible mechanisms may be demethylation of CpG cytosines in the Il1b promoter, which has not been characterized in detail for the mouse. In this study, the methylation level of CpGs located in a region between -3562 and -208 bp upstream of the start of transcription is studied in muscles, bones, liver, thymus, spleen, bone marrow, lymph nodes, lungs, and brain. The methylation level is compared to Il1b expression. Tissue-specific features of CpG methylation are established. It is demonstrated that the region between -2420 and -2406 bp is likely a part of the mouse Il1b promoter/enhancer and may determine the base level of Il1b expression in various tissues. Irradiation at a dose of 6 Gy does not change the methylation profile of most studied CpGs, and therefore, the cause of the stably increased IL1B level after irradiation is unlikely to be a change in the methylation of the studied CpGs in investigated tissues.


Assuntos
Raios gama , Interleucina-1beta/genética , Regiões Promotoras Genéticas/genética , Animais , Osso e Ossos/metabolismo , Osso e Ossos/efeitos da radiação , Ilhas de CpG/genética , Metilação de DNA/efeitos da radiação , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Regiões Promotoras Genéticas/efeitos da radiação , Fatores de Transcrição/metabolismo
16.
Radiat Res ; 193(6): 520-530, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32216710

RESUMO

Genetic and epigenetic profile changes associated with individual radiation sensitivity are well documented and have led to enhanced understanding of the mechanisms of the radiation-induced DNA damage response. However, the search continues to identify reliable biomarkers of individual radiation sensitivity. Herein, we report on a multi-biomarker approach using traditional cytogenetic biomarkers, DNA damage biomarkers and transcriptional microRNA (miR) biomarkers coupled with their potential gene targets to identify radiosensitivity in ataxia-telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines (LCL); ATM-proficient cell lines were used as controls. Cells were 0.05 and 0.5 Gy irradiated, using a linear accelerator, with sham-irradiated cells as controls. At 1 h postirradiation, cells were fixed for γ-H2AX analysis as a measurement of DNA damage, and cytogenetic analysis using the G2 chromosomal sensitivity assay, G-banding and FISH techniques. RNA was also isolated for genetic profiling by microRNA (miR) and RT-PCR analysis. A panel of 752 miR were analyzed, and potential target genes, phosphatase and tensin homolog (PTEN) and cyclin D1 (CCND1), were measured. The cytogenetic assays revealed that although the control cell line had functional cell cycle checkpoints, the radiosensitivity of the control and AT cell lines were similar. Analysis of DNA damage in all cell lines, including an additional control cell line, showed elevated γ-H2AX levels for only one AT cell line. Of the 752 miR analyzed, eight miR were upregulated, and six miR were downregulated in the AT cells compared to the control. Upregulated miR-152-3p, miR-24-5p and miR-92-15p and all downregulated miR were indicated as modulators of PTEN and CCDN1. Further measurement of both genes validated their potential role as radiation-response biomarkers. The multi-biomarker approach not only revealed potential candidates for radiation response, but provided additional mechanistic insights into the response in AT-deficient cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Ciclina D1/metabolismo , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Linfócitos/citologia
17.
Lasers Med Sci ; 35(7): 1509-1518, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32065300

RESUMO

In spinal cord injury (SCI), inflammation is a major mediator of damage and loss of function and is regulated primarily by the bone marrow-derived macrophages (BMDMs). Photobiomodulation (PBM) or low-level light stimulation is known to have anti-inflammatory effects and has previously been used in the treatment of SCI, although its precise cellular mechanisms remain unclear. In the present study, the effect of PBM at 810 nm on classically activated BMDMs was evaluated to investigate the mechanisms underlying its anti-inflammatory effects. BMDMs were cultured and irradiated (810 nm, 2 mW/cm2) following stimulation with lipopolysaccharide and interferon-γ. CCK-8 assay, 2',7'-dichlorofluorescein diacetate assay, and ELISA and western blot analysis were performed to measure cell viability, reactive oxygen species production, and inflammatory marker production, respectively. PBM irradiation of classically activated macrophages significantly increased the cell viability and inhibited reactive oxygen species generation. PBM suppressed the expression of a marker of classically activated macrophages, inducible nitric oxide synthase; decreased the mRNA expression and secretion of pro-inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 beta; and increased the secretion of monocyte chemotactic protein 1. Exposure to PBM likewise significantly reduced the expression and phosphorylation of NF-κB p65 in classically activated BMDMs. Taken together, these results suggest that PBM can successfully modulate inflammation and polarization in classically activated BMDMs. The present study provides a theoretical basis to support wider clinical application of PBM in the treatment of SCI.


Assuntos
Polaridade Celular , Inflamação/radioterapia , Macrófagos/patologia , Animais , Polaridade Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quimiocinas/genética , Quimiocinas/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Ativação de Macrófagos/efeitos da radiação , Macrófagos/efeitos da radiação , Camundongos Endogâmicos BALB C , Fosforilação/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo
18.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054039

RESUMO

Studies of molecular changes occurred in various brain regions after whole-body irradiation showed a significant increase in terms of the importance in gaining insight into how to slow down or prevent the development of long-term side effects such as carcinogenesis, cognitive impairment and other pathologies. We have analyzed nDNA damage and repair, changes in mitochondrial DNA (mtDNA) copy number and in the level of mtDNA heteroplasmy, and also examined changes in the expression of genes involved in the regulation of mitochondrial biogenesis and dynamics in three areas of the rat brain (hippocampus, cortex and cerebellum) after whole-body X-ray irradiation. Long amplicon quantitative polymerase chain reaction (LA-QPCR) was used to detect nDNA and mtDNA damage. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. The mtDNA copy numbers and expression levels of a number of genes were determined by real-time PCR. The results showed that the repair of nDNA damage in the rat brain regions occurs slowly within 24 h; in the hippocampus, this process runs much slower. The number of mtDNA copies in three regions of the rat brain increases with a simultaneous increase in mtDNA heteroplasmy. However, in the hippocampus, the copy number of mutant mtDNAs increases significantly by the time point of 24 h after radiation exposure. Our analysis shows that in the brain regions of irradiated rats, there is a decrease in the expression of genes (ND2, CytB, ATP5O) involved in ATP synthesis, although by the same time point after irradiation, an increase in transcripts of genes regulating mitochondrial biogenesis is observed. On the other hand, analysis of genes that control the dynamics of mitochondria (Mfn1, Fis1) revealed that sharp decrease in gene expression level occurred, only in the hippocampus. Consequently, the structural and functional characteristics of the hippocampus of rats exposed to whole-body radiation can be different, most significantly from those of the other brain regions.


Assuntos
Encéfalo/efeitos da radiação , Núcleo Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Mitocôndrias/efeitos da radiação , Irradiação Corporal Total/efeitos adversos , Animais , Encéfalo/metabolismo , Núcleo Celular/genética , DNA Mitocondrial/genética , Regulação da Expressão Gênica/efeitos da radiação , Genes Mitocondriais/efeitos da radiação , Masculino , Mitocôndrias/genética , Ratos , Ratos Wistar
19.
Exp Mol Pathol ; 114: 104409, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32088192

RESUMO

BACKGROUND: Noise is an important environmental risk factor. Industrial environments are rich in high-intensity infrasound (hi-IFS), which we have found to induce myocardial and coronary perivascular fibrosis in rats. The effects of exposure to IFS on the ventricles have been studied, but not on the atria. We hypothesized that rats exposed to hi-IFS develop atrial remodeling involving fibrosis and connexin 43, which we sought to evaluate. MATERIAL AND METHODS: Seventy-two Wistar rats, half exposed to hi-IFS (120 dB, <20 Hz) during a maximum period of 12 weeks and half age-matched controls, were studied. Atrial fibrosis was analyzed by Chromotrope-aniline blue staining. The immunohistochemical evaluation of Cx43 was performed using the polyclonal antibody connexin-43 m diluted 1:1000 at 4 °C overnight. Digitized images were obtained with an optical microscope using 400× magnifications. The measurements were performed using image J software. A two-way ANOVA model was used to compare the groups. RESULTS: The mean values of the ratio "atrial fibrosis / cardiomyocytes" increased to a maximum of 0.1095 ± 0,04 and 0.5408 ± 0,01, and of the ratio "CX43 / cardiomyocytes" decreased to 0.0834 ± 0,03 and 0.0966 ± 0,03, respectively in IFS-exposed rats and controls. IFS-exposed rats exhibited a significantly higher ratio of fibrosis (p < .001) and lower ratio of Cx43 (p = .009). CONCLUSION: High-intensity infrasound exposure leads to an increase in atrial interstitial fibrosis and a decrease in connexin 43 in rat hearts. This finding reinforces the need for further experimental and clinical studies concerning the effects of exposure to infrasound.


Assuntos
Conexina 43/genética , Fibrose/genética , Coração/fisiopatologia , Ruído/efeitos adversos , Animais , Modelos Animais de Doenças , Fibrose/etiologia , Fibrose/fisiopatologia , Regulação da Expressão Gênica/efeitos da radiação , Coração/efeitos da radiação , Átrios do Coração/fisiopatologia , Átrios do Coração/efeitos da radiação , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Wistar , Fatores de Risco
20.
Lasers Med Sci ; 35(5): 1205-1212, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32030556

RESUMO

The aim of this study was to assess the effects of IL-6 and IL-8 cytokines on human gingival fibroblasts (HGF) cultured in a 3-D model and the possible photobiomodulation (PBM) of such effects by low-level laser therapy. In complete culture medium (DMEM), HGF from a healthy patient were seeded in a type I collagen matrix inserted into 24-well plates. After 5 days of incubation, the cytokines were added or not to serum-free DMEM, which was applied to the cell-enriched matrices. Then, PBM was performed: three consecutive irradiations using LaserTable diode device (780 nm, 0.025 W) at 0.5 J/cm2 were delivered or not to the cells. Twenty-four hours after the last irradiation, cell viability and morphology, gene expression, and synthesis of inflammatory cytokines and growth factors were assessed. The histological evaluation demonstrated that, for all groups, matrices presented homogeneous distribution of cells with elongated morphology. However, numerous cytokine-exposed cells were rounded. IL-6 and IL-8 decreased cell viability, synthesis of VEGF, and gene expression of collagen type I. PBM enhanced cell density in the matrices and stimulated VEGF expression, even after IL-6 challenge. Reduced TNF-α synthesis occurred in those cells subjected to PBM. In conclusion, PBM can penetrate collagen matrix and stimulate HGF, highlighting the relevance of this research model for further phototherapy studies and in vitro biomodulation of the healing process.


Assuntos
Técnicas de Cultura de Células/métodos , Citocinas/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Gengiva/patologia , Inflamação/patologia , Terapia com Luz de Baixa Intensidade , Modelos Biológicos , Sobrevivência Celular/efeitos da radiação , Colágeno Tipo I/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Interleucina-1beta/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Cicatrização/efeitos da radiação
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