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1.
J Photochem Photobiol B ; 203: 111731, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31935633

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic inflammatory disease with a poor prognosis and very few available treatment options. Low-level laser therapy (LLLT) has been gaining prominence as a new and effective anti-inflammatory and immunomodulatory agent. Can lung inflammation and the airway remodeling be regulated by LLLT in an experimental model of IPF in C57Bl/6 mice? The present study investigated if laser attenuates cellular migration to the lungs, the airway remodeling as well as pro-fibrotic cytokines secretion from type II pneumocytes and fibroblasts. Mice were irradiated (780 nm and 30 mW) and then euthanized fifteen days after bleomycin-induced lung fibrosis. Lung inflammation and airway remodeling were evaluated through leukocyte counting in bronchoalveolar lavage fluid (BALF) and analysis of collagen in lung, respectively. Inflammatory cells in blood were also measured. For in vitro assays, bleomycin-activated fibroblasts and type II pneumocytes were irradiated with laser. The pro- and anti-inflammatory cytokines level in BALF as well as cells supernatant were measured by ELISA, and the TGFß in lung was evaluated by flow cytometry. Lung histology was used to analyze collagen fibers around the airways. LLLT reduced both migration of inflammatory cells and deposition of collagen fibers in the lungs. In addition, LLLT downregulated pro-inflammatory cytokines and upregulated the IL-10 secretion from fibroblasts and pneumocytes. Laser therapy greatly reduced total lung TGFß. Systemically, LLLT also reduced the inflammatory cells counted in blood. There is no statistical difference in inflammatory parameters studied between mice of the basal group and the laser-treated mice. Data obtained indicate that laser effectively attenuates the lung inflammation, and the airway remodeling in experimental pulmonary fibrosis is driven to restore the balance between the pro- and anti-inflammatory cytokines in lung and inhibit the pro-fibrotic cytokines secretion from fibroblasts.


Assuntos
Remodelação das Vias Aéreas , Citocinas/metabolismo , Fibrose Pulmonar Idiopática/radioterapia , Lasers , Animais , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Citocinas/análise , Modelos Animais de Doenças , Regulação para Baixo/efeitos da radiação , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Fibrose Pulmonar Idiopática/patologia , Terapia a Laser , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima/efeitos da radiação
2.
J Photochem Photobiol B ; 203: 111738, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31954290

RESUMO

This study aimed to compare the synthesis and secretion of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, and FGF-2 between pulp fibroblasts from human primary teeth (HPF) and stem cell from human deciduous teeth (SHED) before and after photobiomodulation. HPF were obtained from explant technique and characterized by immunohistochemistry, while SHED were obtained from digestion technique and characterized by flow cytometry. HPF (control group) and SHED were plated, let to adhere, and put on serum starvation to synchronize the cell cycles prior to photobiomodulation. Then, both cell lineages were irradiated with 660-nm laser according to the following groups: 2.5 and 3.7 J/cm2. MTT and crystal violet assays respectively verified viability and proliferation. ELISA Multiplex Assay assessed the following proteins: VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, FGF-2, at 6, 12, and 24 h after photobiomodulation, in supernatant and lysate. Two-way ANOVA/Tukey test evaluated cell viability and proliferation, while angiogenic production and secretion values were analyzed by one-way ANOVA (P < .05). Statistically similar HPF and SHED viability and proliferation patterns occurred before and after photobiomodulation (P > .05). HPF exhibited statistically greater values of all angiogenic proteins than did SHED, at all study periods, except for FGF-2 (supernatant; 12 h); VEGFR1 (lysate; non-irradiated; 12 h); and VEGFR1 (lysate; non-irradiated; 24 h). Photobiomodulation changed the synthesis and secretion of angiogenic proteins by HPF. HPF produced and secreted greater values of all tested angiogenic proteins than did SHED before and after irradiation with both energy densities of 2.5 and 3.7 J/cm2.


Assuntos
Fibroblastos/efeitos da radiação , Lasers , Células-Tronco/efeitos da radiação , Linhagem da Célula/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Dente Decíduo/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Food Chem Toxicol ; 136: 110963, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31715308

RESUMO

Excessive exposure to ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes aging of the skin. Most skin diseases caused by UV are attributed to UVB (280-320 nm). The purpose of this study is to investigate the protective effect of diphlorethohydroxycarmalol (DPHC), isolated from the marine brown alga, Ishige okamurae, against UVB-induced photodamage using both in vitro and in vivo models. Results indicate that DPHC remarkably inhibited commercial collagenase and elastase activities. It also reduced intracellular levels of ROS, improved cell viability and collagen content in UVB-irradiated human dermal fibroblasts (HDF cells). In addition, DPHC significantly inhibited activities of intracellular collagenase and elastase and reduced the expression of matrix metalloproteinases (MMPs) and pro-inflammatory cytokines. These events occurred through regulation of nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs) signaling pathways in UVB-irradiated HDF cells. Furthermore, DPHC also protected against in vivo photodamage by decreasing cell death through reducing lipid peroxidation and inflammatory response via decreasing ROS levels in UVB-irradiated zebrafish. In conclusion, DPHC has strong in vitro and in vivo photoprotective effects and has the potential to be used as an ingredient in pharmaceutical and cosmeceutical industries.


Assuntos
Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Feófitas/química , Protetores contra Radiação/uso terapêutico , Envelhecimento da Pele/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Colagenases/metabolismo , Fibroblastos/efeitos da radiação , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Elastase Pancreática/metabolismo , Protetores contra Radiação/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta , Peixe-Zebra
4.
Am J Phys Med Rehabil ; 99(1): 19-25, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31335343

RESUMO

OBJECTIVE: The aim of this study was to analyze the effects of low-intensity pulsed ultrasound therapy under different pulse regimes on cultures of semiconfluent L929 fibroblasts, evaluating cell viability, anatomical structural alterations, modulation of vascular endothelial growth factor, interleukin 6, collagen type 1 alpha 1, collagen type 1 alpha 2, and fibroblast growth factor 7, as well as the amount of inflammatory mediators interleukin 2, interleukin 4, interleukin 6, interferon γ, tumor necrosis factor, interleukin 17A, and interleukin 10 at 24, 48, and 72 hrs. DESIGN: The design was experimental study. METHODS: The treatments consisted of 0.2 W/cm doses at a frequency of 1 MHz, with a pulse rate of 10% and 20%. Viability was assessed by the MTT assay (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide), gene expression by real-time quantitative polymerase chain reaction, and cytokine quantification by flow cytometry. RESULTS: At 48 hrs, ultrasound enhanced cell viability and affected interleukin 6 cytokine production, vascular endothelial growth factor, interleukin 6, type 1 alpha 1 and alpha 2 collagens, and fibroblast growth factor 7 gene modulation. CONCLUSIONS: Low-intensity pulsed ultrasound therapy had a biostimulatory effect on semiconfluent in vitro L929 fibroblast cells, where the group with a dose of 0.2 W/cm-10% (G2) presented higher responses, in all the analyzed aspects, toward the dose pulsed to 20%, confirming its therapeutic properties related to the initial phases of tissue healing.


Assuntos
Anti-Inflamatórios/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Fibroblastos/efeitos da radiação , Terapia por Ultrassom/métodos , Ondas Ultrassônicas , Células Cultivadas , Colágeno Tipo I/efeitos da radiação , Citocinas/efeitos da radiação , Fator 7 de Crescimento de Fibroblastos/efeitos da radiação , Humanos , Mediadores da Inflamação/efeitos da radiação , Interleucina-6/efeitos da radiação , Fator A de Crescimento do Endotélio Vascular/efeitos da radiação
5.
Int J Radiat Biol ; 96(2): 197-205, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31633434

RESUMO

Purpose: To examine the phenomena governing the quantitative relationships between acentric fragments and micronuclei and understand which formulas are useful for curve-fitting of experimental data of micronuclei.Materials and methods: A stochastic model, including the phenomena of inclusion, coalescence and culling out, was developed and applied to experimental data.Results: Probabilities for inclusion/exclusion of acentric fragments into daughter nuclei and for coalescence of many fragments into a single micronucleus were found to be not cell type-specific. The biological basis for this result is explained with the lack of DNA damage checkpoints between metaphase (when acentric fragments are scored) and telophase (when micronuclei are formed). The phenomenon of "culling out" cells with high numbers of acentric fragments is also described, along with its proposed biological mechanism.Conclusions: Apart from complex formulas that describe these phenomena, we discuss which simple formulas can best approximate them and when is the case to use them for curve fitting of micronuclei data.


Assuntos
Núcleo Celular/metabolismo , Aberrações Cromossômicas/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Testes para Micronúcleos , Apoptose , Dano ao DNA , Relação Dose-Resposta à Radiação , Células Epiteliais/efeitos da radiação , Fibroblastos/efeitos da radiação , Humanos , Cinética , Modelos Lineares , Metáfase , Probabilidade , Telófase
6.
Int J Radiat Biol ; 96(2): 179-186, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31633439

RESUMO

Purpose: We studied lethal and mutagenic bystander effects in normal human fibroblasts irradiated with low-energy-carbon ions.Materials and methods: After cells reached confluence, cells were irradiated with initial energies of 6 MeV/n carbon ions. The residual energy and LET value were 4.6 MeV/n and 309 keV/µm. The doses used for survival and mutational studies were 0.082 and 0.16 Gy. Irradiation was carried out using 4 different irradiation conditions and plating conditions: (1) The entire cell area on the Mylar film was irradiated (We abbreviate as 'all irradiation'); (2) Irradiated and unirradiated cells were pooled in a 1:1 ratio and plated as a single culture until the plating for lethal and mutagenic experiments (We abbreviate as 'mixed population'); (3) Only half of the area on the Mylar film were irradiated using an ion-beam stopper (We abbreviate as 'half irradiation'); and (4) Only half of the area of the cells were irradiated, and a specific inhibitor of gap junctions was added to the culture (We abbreviate as 'half irradiation with inhibitor'). Cell samples were analyzed for lethal and mutagenic bystander effects, including a PCR evaluation of the mutation spectrum.Results: The surviving fraction of all irradiation was the same as the half irradiation case. The surviving fractions of both mixed population and the half irradiation with inhibitor were the same level and higher than those of all irradiation and half irradiation. The mutation frequencies at the HPRT (the hypoxanthine-guanine phosphoribosyl transferase) locus of all irradiation and half irradiation were at the same level and were higher than those of mixed population and half irradiation with inhibitor, respectively.Conclusion: There is evidence that the bystander effects for both lethality and mutagenicity occurred in the unirradiated half of the cells, in which only half of the cells were irradiated with the carbon ions. These results suggest that the bystander cellular effects via gap-junction-mediated cell-cell communication are induced by high-LET-carbon ions.


Assuntos
Efeito Espectador/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Fibroblastos/efeitos da radiação , Junções Comunicantes/efeitos da radiação , Radioterapia com Íons Pesados/métodos , Mutagênese , Carbono/química , Técnicas de Cultura de Células , Dano ao DNA , Relação Dose-Resposta à Radiação , Íons Pesados , Humanos , Hipoxantina Fosforribosiltransferase/genética , Íons , Transferência Linear de Energia , Mutagênicos , Mutação
7.
Nucleic Acids Res ; 48(3): 1314-1326, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31822909

RESUMO

Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.


Assuntos
Sobrevivência Celular/genética , Neoplasias da Próstata/radioterapia , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Sobrevivência Celular/efeitos da radiação , Cromatina/efeitos da radiação , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Fibroblastos/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Masculino , Camundongos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos da radiação
8.
Photochem Photobiol Sci ; 19(1): 40-48, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31776533

RESUMO

Although the dichlorofluorescein (DCF) assay is widely used to detect the production of UVA-induced ROS, the photostability and phototoxicity of the probe after UVA irradiation remains controversial and the experimental conditions often vary across studies, making it difficult to compare results from different studies. This study aimed to evaluate the suitability of the DCF assay for detection of UVA-induced ROS in human cells after UVA irradiation. Human primary fibroblasts (HPF) and HaCaT cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) (2, 10, and 50 µM) for 10 and 30 min, before and after exposure to UVA radiation (5-50 J cm-2). Fluorescence was recorded immediately or 30 min after irradiation using three different techniques: microplate reading, flow cytometry, and confocal scanning microscopy. Cell viability was assessed by flow cytometry before and after UVA exposure. A UVA-dose-dependent increase in ROS was observed at 5-50 µM DCFDA, and the magnitude of the fluorescent signal was affected by RPMI medium, as well as DCFDA loading concentration and incubation period. However, higher concentrations of DCFDA compromised the viability of both HaCaT and HPF cells after UVA irradiation. The most sensitive and reliable combination for the ROS assay was pre-incubation with 10 µM DCFDA for 30 min in PBS. Reading the fluorescence 30 min after UVA irradiation diminished the emission signal, as did the DCFDA post-incubation. In conclusion, this single-point DCF assay allowed reproducible and sensitive UVA-induced ROS detection in HaCaT and HPF cells without compromising the cell viability or morphology.


Assuntos
Fibroblastos/efeitos da radiação , Fluoresceínas/farmacologia , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Raios Ultravioleta , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Fluoresceínas/química , Humanos , Processos Fotoquímicos/efeitos da radiação , Relação Estrutura-Atividade
9.
Radiat Res ; 193(1): 63-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31714866

RESUMO

It is well known that mitochondria and the endoplasmic reticulum (ER) play important roles in radiation response, but their functions in radiation-induced bystander effect (RIBE) are largely unclear. In this study, we found that when a small portion of cells in a population of human lung fibroblast MRC-5 cells were precisely irradiated through either the nuclei or cytoplasm with counted microbeam protons, the yield of micronuclei (MN) and the levels of intracellular reactive oxygen species (ROS) in nonirradiated cells neighboring irradiated cells were significantly increased. Mito/ER-tracker staining demonstrated that the mitochondria were clearly activated after nuclear irradiation and ER mass approached a higher level after cytoplasmic irradiation. Moreover, the radiation-induced ROS was diminished by rotenone, an inhibitor of mitochondria activation, but it was not influenced by siRNA interference of BiP, an ER regulation protein. While for nuclear irradiation, rotenone-enhanced radiation-induced ER expression, and BiP siRNA eliminated radiation-induced activation of mitochondria, these phenomena were not observed for cytoplasmic irradiation. Bystander MN was reduced by rotenone but enhanced by BiP siRNA. When the cells were treated with both rotenone and BiP siRNA, the MN yield was reduced for nuclear irradiation but was enhanced for cytoplasmic irradiation. Our results suggest that the organelles of mitochondria and ER have different roles in RIBE with respect to nuclear and cytoplasmic irradiation, and the function of ER is a prerequisite for mitochondrial activation.


Assuntos
Efeito Espectador/efeitos da radiação , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos da radiação , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Prótons/efeitos adversos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Humanos , Espécies Reativas de Oxigênio/metabolismo
10.
Nucleic Acids Res ; 48(1): 231-248, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31722399

RESUMO

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição Genética , Ubiquitina/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Raios Ultravioleta
11.
J Photochem Photobiol B ; 202: 111720, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31841988

RESUMO

It has been widely reported that ultraviolet-B (UV-B) radiation is the main extrinsic etiological agent that causes skin photodamage. UV-B exposure mediated photodamage (photo-aging/photo-carcinogenesis) to human skin is caused due to several physiological events at tissue, cellular and molecular levels that lead to impairment of skin function and integrity. In the present study, we investigated the protective role of Trigonelline (TG) against UV-B induced photo-damage in Human Dermal Fibroblasts (Hs68 cells) and Balb/C mice. We exposed human skin fibroblasts and Balb/C mice to UV-B radiation and evaluated various parameters of cellular damage, including, oxidative stress, cytosolic calcium (Ca2+) levels, apoptotic and ER-stress marker proteins. We found that UV-B irradiation induced ROS generation lead to the depletion of endoplasmic reticulum (ER) calcium and increased the expression of ER stress protein markers (phosphorylated elf2α, CHOP, ATF4) as well as apoptotic protein markers (Bcl2, Bax and caspase-9) in a dose and time dependent manner in Hs68 cells. We then determined the effect of TG treatment on UV-B -induced cell death in Hs68 cells and observed that cells exposed to UV-B radiation and treated with TG had a significantly higher survival rate compared to cells exposed to UV-B radiation alone. TG treatment successfully reduced oxidative stress; restored Ca2+ homeostasis and re-established the ER function and prevented apoptotic cell death process. Our results suggest that TG can be used as a potential therapeutic/cosmeceutic agent in preventing skin photo-damage.


Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos da radiação , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
12.
Arch Oral Biol ; 109: 104557, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31557575

RESUMO

OBJECTIVE: To investigate the effects of dental x-ray on proliferation and mineralization in human primary osteoblasts as well as on proliferation and apoptotic potential in human periodontal ligament (PDL) cells. DESIGN: Primary osteoblasts and PDL cells were irradiated with various doses of periapical radiography by repeated exposures and further incubated for 1, 3 or 7 days. Cell proliferation was assayed by BrdU incorporation. The effect of dental x-ray on mineralization in osteoblasts either before or after x-ray exposures was determined by Alizarin red staining. Both mRNA and protein expressions of BCL-2, an anti-apoptotic gene, and BAX, a pro-apoptotic gene, in PDL cells were analyzed by RT-qPCR and immunoblotting analysis, respectively. RESULTS: Neither the proliferative nor the mineralization ability of irradiated osteoblasts was different from that of non-irradiated osteoblasts at any doses or time points. By contrast, there was a significant decrease in the proliferation of PDL cells on day 3 after repeated exposures to dental x-ray for 20 times (P < 0.05), whereas the ratio of BCL-2 to BAX mRNA and protein expressions in these irradiated PDL cells was significantly increased (P < 0.05). CONCLUSIONS: Upon multiple exposures to dental x-ray used in intraoral radiography up to 20 times, there is no effect on the proliferation or the mineralization of osteoblasts, whereas the proliferative and apoptotic potentials of PDL cells are transiently decreased.


Assuntos
Fibroblastos/efeitos da radiação , Osteoblastos/efeitos da radiação , Ligamento Periodontal/citologia , Raios X , Adolescente , Adulto , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Criança , Feminino , Humanos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/genética , Radiografia Dentária , Adulto Jovem , Proteína X Associada a bcl-2/genética
13.
J Sci Food Agric ; 100(2): 672-681, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31583701

RESUMO

BACKGROUND: Hibiscus sabdariffa is commonly used in daily life and its extract is applied widely in food and cosmetics. However, it has not been evaluated for its anti-aging effects. RESULTS: Hibiscus sabdariffa calyx aqueous extract (HSCAE) has shown potential collagenase activity suppression effects, together with tyrosinase activity inhibition, and anti-oxidation as a free radical scavenger. The current investigation demonstrated that HSCAE was not cytotoxic in skin fibroblasts, and it significantly decreased ultraviolet B (UVB)-induced reactive oxygen species (ROS) on a flow cytometry assay. Moreover, HSCAE reduced matrix metalloproteinase (MMP) expression, increased tissue inhibition of metalloproteinase (TIMP)-1 level, and enhanced collagen content by inhibiting collagenase activity. It also blocked mRNA and protein expressions of melanin production pathway key factors, including the microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and dopachrome tautomerase-2 (TRP-2). CONCLUSION: These results demonstrated, for the first time, the potential of HSCAE as a natural antioxidant with the ability to maintain collagen production and to decrease melanin syntheses under UVB radiation, for anti-aging effects. © 2019 Society of Chemical Industry.


Assuntos
Depuradores de Radicais Livres/farmacologia , Hibiscus/química , Extratos Vegetais/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Animais , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Camundongos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos
14.
Int J Mol Sci ; 21(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861704

RESUMO

Human adipose-derived mesenchymal stem cells-conditioned medium (ADSC-CM) contains cytokines and growth factors that can facilitate the regeneration and repair of various tissues and organs. In the present study, the protective activity of ADSC-CM treatment was investigated in UVB-irradiated human keratinocyte cell line HaCaTs and normal human dermal fibroblasts (NHDFs). It was found that ADSC-CM can modulate the expression of the signaling molecules in the early UVB responsive signaling pathways, including mitogen activated protein kinases (MAPKs), activator protein 1 (AP-1), and nuclear factor kappa B (NF-κB). In addition, ADSC-CM treatment could upregulate antioxidant response element (ARE) such as phase II gene heme oxygenase-1 (HO-1) and increase the expression of collagen synthesis enhancer gene transforming growth factor-ß (TGF-ß). The expression of matrix metalloproteinase-1 (MMP-1) and procollagen type I synthesis inhibitors such as interleukin-6 (IL-6) was also found to be suppressed upon ADSC-CM treatment. Taken together, our study illustrates the anti-photoaging activities of ADSC-CM in cell-based models.


Assuntos
Tecido Adiposo/citologia , Meios de Cultivo Condicionados/farmacologia , Queratinócitos/citologia , Células-Tronco Mesenquimais/citologia , Envelhecimento da Pele/efeitos dos fármacos , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Metaloproteinase 1 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta/efeitos adversos
15.
Proc Natl Acad Sci U S A ; 116(48): 24196-24205, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31723047

RESUMO

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at A2-15TTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers.


Assuntos
Fibroblastos/efeitos da radiação , Genoma Humano/efeitos da radiação , Melanócitos/efeitos da radiação , Nucleotídeos de Pirimidina/efeitos da radiação , Regiões 5' não Traduzidas , Células Cultivadas , Dano ao DNA/efeitos da radiação , Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Melanócitos/fisiologia , Melanoma/genética , Mutação , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Dímeros de Pirimidina/efeitos da radiação , Neoplasias Cutâneas/genética , Serina-Treonina Quinases TOR/genética , Raios Ultravioleta
16.
Mutat Res ; 847: 503105, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31699341

RESUMO

18F-FDG PET/CT imaging is used in the diagnosis of diseases, including cancers. The principal photons used for imaging are 511 ke V gamma photons resulting from positron annihilation. The absorbed dose varies among body organs, depending on administered radioactivity and biological clearance. We have attempted to evaluate DNA double-strand breaks (DSB) and toxicity induced in V79 lung fibroblast cells in vitro by 18F-FDG, at doses which might result from PET procedures. Cells were irradiated by 18F-FDG at doses (14.51 and 26.86 mGy), comparable to absorbed doses received by critical organs during PET procedures. The biological endpoints measured were formation of γ-H2AX foci, mitochondrial stress, chromosomal aberrations, and cell cycle perturbation. Irradiation induced DSB (γH2AX assay), mitochondrial depolarization, and both chromosome and chromatid types of aberrations. At higher radiation doses, increased aneuploidy and reduced mitotic activity were also seen. Thus, significant biological effects were observed at the doses delivered by the 18F-FDG exposure and the effects increased with dose.


Assuntos
Aberrações Cromossômicas , Dano ao DNA , Fibroblastos/efeitos da radiação , Radioisótopos de Flúor/toxicidade , Fluordesoxiglucose F18/toxicidade , Raios gama/efeitos adversos , Compostos Radiofarmacêuticos/toxicidade , Aneuploidia , Animais , Benzimidazóis , Carbocianinas , Ciclo Celular/efeitos da radiação , Linhagem Celular , Cromátides/efeitos da radiação , Cromátides/ultraestrutura , Cromossomos/efeitos da radiação , Cromossomos/ultraestrutura , Cricetulus , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Relação Dose-Resposta à Radiação , Fibroblastos/ultraestrutura , Histonas/genética , Cariotipagem , Pulmão/citologia , Masculino , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitose/efeitos da radiação
17.
J Radiat Res ; 60(6): 719-728, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31665364

RESUMO

Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a dual capability to self-renew and differentiate into all cell types necessary to develop an entire organism. Differentiation is associated with dynamic epigenetic alteration and transcriptional change, while self-renewal depends on maintaining the genome DNA accurately. Genome stability of PSCs is strictly regulated to maintain pluripotency. However, the DNA damage response (DDR) mechanism in PSCs is still unclear. There is accumulating evidence that genome stability and pluripotency are regulated by a transcriptional change in undifferentiated and differentiated states. iPSCs are ideal for analyzing transcriptional regulation during reprogramming and differentiation. This study aimed to elucidate the transcriptional alteration surrounding genome stability maintenance, including DNA repair, cell cycle checkpoints and apoptosis in fibroblasts, iPSCs and neural progenitor cells (NPCs) derived from iPSCs as differentiated cells. After ionizing radiation exposure, foci for the DNA double-stranded break marker γ-H2AX increased, peaking at 0.5 h in all cells (>90%), decreasing after 4 h in fibroblasts (32.3%) and NPCs (22.3%), but still remaining at 52.5% (NB1RGB C2 clone) and 54.7% (201B7 cells) in iPSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were detected, indicating that iPSCs' apoptosis increases. In addition, RNA sequencing (RNA-Seq) analysis showed high expression of apoptosis genes (TP53, CASP3 and BID) in iPSCs. Results suggested that increased apoptosis activity maintains accurate, undifferentiated genome DNA in the cell population.


Assuntos
Apoptose/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , Dano ao DNA/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcrição Genética , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Reprogramação Celular/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos da radiação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos da radiação , Radiação Ionizante , Pele/citologia
18.
In Vivo ; 33(6): 1773-1784, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662502

RESUMO

BACKGROUND/AIM: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-ß-secreting M2 macrophages in association with ionizing radiation-induced EMT. MATERIALS AND METHODS: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. RESULTS: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1ß, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-ß and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-ß. Moreover, the elimination of TGF-ß from M2 macrophages attenuated mesenchymal transition of MLE12. CONCLUSION: TGF-ß-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.


Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos da radiação , Pulmão/metabolismo , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Pulmão/efeitos da radiação , Macrófagos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Pneumonite por Radiação/etiologia , Pneumonite por Radiação/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos da radiação
19.
Nutrients ; 11(10)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614689

RESUMO

Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.


Assuntos
Fibroblastos/efeitos dos fármacos , Lactobacillus plantarum/metabolismo , Extratos Vegetais/farmacologia , Rubus/química , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Fermentação , Fibroblastos/efeitos da radiação , Prepúcio do Pênis/citologia , Frutas/química , Masculino , Camundongos , Camundongos Pelados , Extratos Vegetais/química , Envelhecimento da Pele/efeitos da radiação
20.
Int J Mol Sci ; 20(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627284

RESUMO

Rapidly evolving laser technologies have led to the development of laser-generated particle accelerators as an alternative to conventional facilities. However, the radiobiological characteristics need to be determined to enhance their applications in biology and medicine. In this study, the radiobiological effects of ultrashort pulsed electron beam (UPEB) and X-ray radiation in human lung fibroblasts (MRC-5 cell line) exposed to doses of 0.1, 0.5, and 1 Gy are compared. The changes of γH2AX foci number as a marker of DNA double-strand breaks (DSBs) were analyzed. In addition, the micronuclei induction and cell death via apoptosis were studied. We found that the biological action of UPEB-radiation compared to X-rays was characterized by significantly slower γH2AX foci elimination (with a dose of 1 Gy) and strong apoptosis induction (with doses of 0.5 and 1.0 Gy), accompanied by a slight increase in micronuclei formation (dose of 1 Gy). Our data suggest that UPEB radiation produces more complex DNA damage than X-ray radiation, leading to cell death rather than cytogenetic disturbance.


Assuntos
Apoptose/efeitos da radiação , Fibroblastos/efeitos da radiação , Terapia a Laser , Lasers , Pulmão/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Histonas/genética , Humanos , Testes para Micronúcleos
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