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Periodontal ligament (PDL) cells play an important role in mechanosensing and secretion of signaling molecules during bone remodeling. However, the regulatory mechanism is unknown. The aim of the present study is to investigate the expression pattern of periostin and sclerostin in response to orthodontic forces in periodontal ligament cells in vitro. PDL cells were isolated from extracted teeth and treated with compressive forces of 25 gr/cm2 or equiaxial tension forces at frequency 1 Hz for 0, 24, 48, and 72 h. qRT-PCR was applied to evaluate the gene expressions. The secretion of sclerostin and periostin was assessed using ELISA. DAPI staining was used to evaluate apoptosis. The expression of sclerostin elevated significantly at protein and gene levels under compression forces after 24 h, while the application of tensile forces induced the expression of periostin and its upstream regulator RUNX2 (p < 0.05). Gene expression up-regulation was significant for POSTN and RUNX2 after 48 and 72 h tensile forces. Also, the gene expression of sclerostin reduced in a time-dependent manner after application of tensile force. The compression forces enhanced apoptosis to 7.5 ± 3.5% and induced gene expression of apoptotic markers of CASP9, and BCL2 within 72 h of exposure. Periostin and sclerostin play an important role in orthodontic loads and their expressions are affected oppositely by compressive and tensile forces that might be suggested as a biomarker for assessment of bone remodeling during orthodontic treatment.
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Subunidade alfa 1 de Fator de Ligação ao Core , Ligamento Periodontal , Humanos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Remodelação Óssea , Biomarcadores , Pressão , Estresse Mecânico , Técnicas de Movimentação Dentária , Células Cultivadas , Moléculas de Adesão Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Patients with diabetes mellitus (DM) suffer from oral complications related to oral infections, periodontal diseases, and endodontic lesions. Emerging evidence has revealed the contribution of the epigenetic process as the underlying mechanism of DM complications. DNA methylation, histone modifications, and non-coding RNAs are epigenetic regulators that directly affect gene expression. The present review elaborated on the role of epigenetic dysregulation in the etiology of diabetes-related periodontal and endodontic diseases. The narrative review study was prepared using databases such as PubMed, Google Scholar, Science Direct, and Scopus. The formation of glycation products as a result of hyperglycemic condition increases oxidative stress, and elevates chronic inflammatory mediators that could in turn adversely change the cellular environment and alter the epigenetic status. This process contributes to the alteration of regulatory genes expression, leading to the development of diabetes-induced bone complications and impaired odontogenic capacity of pulp. Indeed, epigenetic mechanisms mediate the interaction between gene expression and DM cellular environment. Further investigations on epigenetic factors involved in DM oral complications may provide novel therapeutic targets.
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Complicações do Diabetes , Diabetes Mellitus , Hiperglicemia , Humanos , Epigênese Genética , Metilação de DNA/genética , Complicações do Diabetes/genética , Hiperglicemia/genética , Diabetes Mellitus/genéticaRESUMO
An in vitro study was designed to evaluate the effects of photobiomodulation (PBM) with 915-nm diode laser on human gingival fibroblast (HGF) cells under hyperglycemic condition. The HGF cells were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 30 mM glucose concentration for 48 h to mimic the hyperglycemic condition. Subsequently, the cells received three sessions of PBM (915 nm, continuous emission mode, 200 mW, energy density values of 3.2, 6, and 9.2 J/cm2). Twenty-four hours post-irradiation, cell proliferation, expression of interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) were assessed with MTT and real-time polymerase chain reaction (PCR) tests, respectively. Also, reactive oxygen species (ROS) production was measured using CM-H2DCFDA fluorimetry. No changes were detected in the cell proliferation rate between the high glucose control group and laser-treated cells, while VEGF and IL-6 gene expression levels increased significantly after PBM in the high glucose-treated cells group. ROS level was significantly decreased in the irradiated cells in high-glucose medium compared with the high glucose control group. Our study revealed the inductive role of 915-nm-mediated PBM on VEGF and the inflammatory response while concurrently reducing reactive oxygen species production in HGF cells in hyperglycemic conditions.
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Interleucina-6 , Terapia com Luz de Baixa Intensidade , Humanos , Interleucina-6/genética , Fator A de Crescimento do Endotélio Vascular/genética , Espécies Reativas de Oxigênio/metabolismo , Glicemia , Fibroblastos/efeitos da radiação , Células CultivadasRESUMO
Environmental toxicants can regulate gene expression in the absence of DNA mutations via epigenetic mechanisms such as DNA methylation, histone modifications, and non-coding RNAs' (ncRNAs). Here, all three epigenetic modifications for seven important categories of diseases and the impact of eleven main environmental factors on epigenetic modifications were discussed. Epigenetic-related mechanisms are among the factors that could explain the root cause of a wide range of common diseases. Its overall impression on the development of diseases can help us diagnose and treat diseases, and besides, predict transgenerational and intergenerational effects. This comprehensive article attempted to address the relationship between environmental factors and epigenetic modifications that cause diseases in different categories. The studies main gap is that the precise role of environmentally-induced epigenetic alterations in the etiology of the disorders is unknown; thus, still more well-designed researches need to be accomplished to fill this gap. The present review aimed to first summarize the adverse effect of certain chemicals on the epigenome that may involve in the onset of particular disease based on in vitro and in vivo models. Subsequently, the possible adverse epigenetic changes that can lead to many human diseases were discussed.
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Metilação de DNA , Epigênese Genética , Humanos , DNA , Substâncias PerigosasRESUMO
Endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), which is widely used in the plastic industry, have recently been considered to be involved in the pathogenesis of metabolic disorders, including obesity and diabetes. The present study aimed to examine the potentially detrimental effects of BPA on glucose and energy metabolism at the epigenetic level. The blood glucose profile of Wistar rats receiving different oral doses of BPA over 28 days was assessed. At the end of the treatment, the islets of Langerhans were isolated and purified, and their RNA content was extracted. MicroRNA (miRNA) profiling was evaluated using the next generation sequencing (NGS) method. After performing bioinformatic analysis of the NGS data, the gene ontology and data enrichment in terms of significantly disturbed miRNAs were evaluated through different databases, including Enrichr and DIANA tools. Additionally, the DNA methylation and the level of expression of two critical genes in glucose metabolism (PPARγ, Pdx1) were assessed. Subchronic BPA exposure (406 mg/kg/day) disturbed the blood glucose profile (fasting blood glucose and oral glucose tolerance) of Wistar rats and resulted in considerable cytotoxicity. NGS data analyses revealed that the expression of some crucial miRNAs involved in ß-cell metabolism and diabetes occurrence and development, including miR-375, miR-676, miR-126-a, and miR-340-5p, was significantly disrupted. According to the DNA methylation evaluation, both PPARγ and Pdx1 genes underwent changes in the methylation level at particular loci on the gene's promoter. The expression levels of these genes were upregulated and downregulated, respectively. Overall, subchronic BPA exposure could cause epigenetic dysregulation at the gene level and interfere with the expression of key miRNAs and the methylation process of genes involved in glucose homeostasis. Understanding the exact underlying mechanisms by which BPA and other EDCs induce endocrine disturbance could be of great importance in the way of finding new preventive and therapeutic approaches.
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Compostos Benzidrílicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Fenóis/farmacologia , Administração Oral , Animais , Compostos Benzidrílicos/administração & dosagem , Biologia Computacional , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Epigênese Genética/genética , Ilhotas Pancreáticas/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Fenóis/administração & dosagem , Ratos , Ratos WistarRESUMO
Extended exposure to inorganic arsenic through contaminated drinking water has been linked with increased incidence of diabetes mellitus. The most common exposure occurs through the consumption of contaminated drinking water mainly through geogenic sources of inorganic arsenic. Epigenetic modifications are important mechanisms through which environmental pollutants could exert their toxic effects. Bisulfite sequencing polymerase chain reaction method followed by Sanger sequencing was performed for DNA methylation analysis. Our results showed that sodium arsenite treatment significantly reduced insulin secretion in pancreatic islets. It was revealed that the methylation of glucose transporter 2 (Glut2) gene was changed at two cytosine-phosphate-guanine (CpG) sites (-1743, -1734) in the promoter region of the sodium arsenite-treated group comparing to the control. No changes were observed in the methylation status of peroxisome proliferator-activated receptor-gamma (PPARγ), pancreatic and duodenal homeobox 1 (Pdx1) and insulin 2 (Ins2) CpG sites in the targeted regions. Measuring the gene expression level showed increase in Glut2 expression, while the expression of insulin (INS) and Pdx1 were significantly affected by sodium arsenite treatment. This study revealed that exposure to sodium arsenite changed the DNA methylation pattern of Glut2, a key transporter of glucose entry into the pancreatic beta cells (ß-cells). Our data suggested possible epigenetic-mediated toxicity mechanism for arsenite-induced ß-cells dysfunction. Further studies are needed to dissect the precise epigenetic modulatory activity of sodium arsenite that affect the biogenesis of insulin.
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Arsenitos/toxicidade , Metilação de DNA/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Compostos de Sódio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Epigênese Genética/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Insulina/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Transativadores/genéticaRESUMO
Epigenotoxicology is an emerging field of study that investigates the non-genotoxic epigenetic effects of environmental toxicants resulting in alteration of normal gene expression and disruption of cell function. Recent findings on the role of toxicant-induced epigenetic modifications in the development of degenerative diseases have opened up a promising research direction to explore epigenetic therapy approaches and related prognostic biomarkers. In this review, we presented comprehensive data on epigenetic alterations identified in various diseases, including cancer, autoimmune disorders, pulmonary conditions as well as cardiovascular, gastrointestinal and bone disease. Although data on abnormalities of DNA methylation and their role in the development of diseases are abundant, less is known about the impact of histone modifications and microRNA expressions. Further, we discussed the effects of selected common environmental toxicants on epigenetic modifications and their association with particular abnormalities. A number of different environmental toxicants have been identified for their role in aberrant DNA methylation, histone modifications, and microRNA expression. Such epigenetic effects were shown to be tissue-type specific and highly associated with the level and duration of exposure. Finally, we described present and future therapeutic strategies, including medicines and dietary compounds for combating the toxicant-induced epigenetic alterations. There are currently seven histone deacetylase inhibitors and two DNA methyltransferase inhibitors approved for clinical use and many other promising candidates are in preclinical and clinical testing. Dietary compounds are thought to be the effective and safe strategies for treating and prevention of epigenetic pathophysiological conditions. Still more concentrated epigenetic researches are required for evaluation of chemical toxicity and identifying the causal association between key epigenetic alteration and disease.
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Exposição Ambiental/efeitos adversos , Poluentes Ambientais/toxicidade , Epigênese Genética/efeitos dos fármacos , Doenças Autoimunes/epidemiologia , Doenças Autoimunes/genética , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Metilação de DNA/efeitos dos fármacos , Dieta , Inibidores de Histona Desacetilases/farmacologia , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias/epidemiologia , Neoplasias/genéticaRESUMO
Ethephon is one of the most widely used plant growth regulator in agriculture that its application has been increased in recent years. Many reports have raised concern over the safety of this organophosphorus compound. The aim of the current study was to assess the potential genotoxic effect of ethephon on murine embryonic fibroblast (MEF) cell line, using two genotoxicity endpoints: γH2AX expression and comet assay. γH2AX served as an early and sensitive biomarker of genotoxic damage. Oxidative stress biomarkers, including reactive oxygen species (ROS), lipid peroxidation (LPO) and total antioxidant capacity were also examined. The results showed a significant increase in cell proliferation, 24 h post-treatment with 10, 40,160 µg/ml ethephon, while at the higher concentrations cytotoxic effect was observed. The γH2AX expression and γH2AX foci count per cell were significantly increased at non-cytotoxic concentrations of ethephon, accompanied with increased DNA damage as illustrated by comet assay. LPO and ROS levels were elevated only at 160 µg/ml and higher doses. The results interestingly showed that low non-cytotoxic doses of ethephon promoted DNA damage inducing cell proliferation, raising the possibility of ethephon mutagenicity. The genotoxic effect of ethephon at low doses might not relate to oxidative damage and that increased in the level of ROS and LPO generation at higher doses could account for the cytotoxic effect of ethephon. Taken together, our study provides strong in vitro evidence on potential genotoxicity of ethephon at low doses. More precise studies are needed to clarify the mutagenic effect of chronic exposure to ethephon.
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Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Fibroblastos/efeitos dos fármacos , Histonas/genética , Mutagênicos/toxicidade , Compostos Organofosforados/toxicidade , Animais , Proliferação de Células/genética , Ensaio Cometa , Fibroblastos/metabolismo , Citometria de Fluxo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Espécies Reativas de Oxigênio/metabolismoRESUMO
Animal-based test systems have traditionally been used to screen for the potential teratogenic activity of drugs. Still, their deficits in predicting precise human-specific outcomes and ethical concerns have led to a need for alternative approaches. In vitro, teratogenicity testing using cell cultures or other in vitro systems is a potential alternative. Of the different in vitro platforms, the mouse embryonic stem cell test (mEST) is currently the most widely used and validated in vitro test for assessing the potential effects of teratogens on early embryonic development. The mEST involves exposing mouse embryonic stem cells to the test compound and monitoring their differentiation for several days.Nevertheless, its predictive ability was comparatively lower when distinguishing weak developmental toxicants from non-toxic substances. Since then, several modifications and adaptations of the mEST protocol have been developed. This chapter describes an alternative method based on molecular approaches to predict embryotoxicity. This method, originated from the mEST, analyzes the expression of differentiation genes involved in the development of mesoderm, endoderm, and stoderm and allows screening embryo-toxicants with different mechanisms of action. The hanging drops embryoid bodies used in the original mEST protocol have been replaced with monolayer culture, and thus the process has been shortened. In general, the method shows higher predictability compared with the traditional ones.
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Células-Tronco Embrionárias , Teratogênese , Feminino , Gravidez , Humanos , Animais , Camundongos , Células-Tronco Embrionárias Murinas , Teratogênicos/toxicidade , Corpos Embrioides , Substâncias PerigosasRESUMO
This study evaluated the effect of photobiomodulation therapy (PBMT) using 660 and 808 nm diode lasers (individual and in combination) on periodontal ligament mesenchymal stem cells (PDLSCs) in the presence of zoledronic acid (ZA). PDLSCs were cultured for 48 h in DMEM complete medium containing 5 µM ZA. PBMT was done three times with a 24-h interval in groups 1 (660 nm, 5 J/cm2 ), 2 (880 nm, 3 J/cm2 ), and 3 (660 + 808 nm) either in normal or ZA-treated culture medium. Control groups did not receive PBMT. Twenty-four hours post-irradiation, cell proliferation and expression of RANKL and OPG were assessed using MTT and real-time PCR tests, respectively. The results showed a significant decrease in cell viability in ZA-treated cells (p < 0.001). Additionally, ZA induced the expression of OPG (p = 0.03) while reducing RANKL (p < 0.001). Cell proliferation was significantly increased in 808 and 660 + 808 nm groups. Moreover, all PBMT groups could significantly increase and decrease the RANKL and OPG, respectively, in the presence of ZA (all p < 0.001). A combination of 660 + 808 nm showed the highest effects on both genes. In conclusion, it seems that PBMT can modulate the effects of ZA by inducing PDLSC proliferation and increasing RANKL-to-OPG gene expression ratio.
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BACKGROUND AND AIM: Regenerative endodontic procedures (REPs) are oriented by the principles of tissue engineering, incorporating dental pulp stem cells (DPSC), crucial growth factors like Transforming growth factor-ß (TGF-ß1), and scaffolds to facilitate the regeneration of dental pulp tissues. The present study aimed to investigate the effect of photobiomodulation (PBM) therapy, using an 808 nm diode laser on cellular modulation mechanisms in REPs. METHOD AND MATERIAL: A total of 108 human dentin discs obtained from intact single root teeth were randomly assigned into six groups (n = 8): 1. Positive control (EDTA), 2. PBM-1 (3 J/cm2), 3. PBM-2 (5 J/cm2), 4. EDTA+PBM-1, 5. EDTA+PBM-2, and 6. Negative control (NaOCl). Then, an extract solution was prepared from each disc and the concentration of released TGF-ß1 from the discs was measured using enzyme-linked immunosorbent assay (ELISA). Moreover, the extract solution was added to DPSC culture medium to evaluate cell viability and migration through MTT assay and scratch test, respectively. RESULT: The group exposed to PBM-1 showed the highest cell viability, while treatment with EDTA and EDTA+PBM-2 decreased cellular viability. Also, the PBM-treated groups showed significantly higher release of TGF-ß1 compared to the negative control. EDTA and EDTA+PBM-1 showed the highest release among all the groups. No significant difference was found between EDTA and EDTA+PBM-1, as well as between PBM-1 and PBM-2. Moreover, the PBM-1 group exhibited the highest migration after 24 h, which was significantly greater than other groups, except for the PBM-2 group. CONCLUSION: According to the obtained data, 808 nm mediated-PBM (3 J/cm2), both independently and in conjunction with EDTA, enhanced the release of TGF-ß1 from dentin and improved cell viability and migration of DPSCs. It seems that, PBM under the specific parameters employed in this study, could be an effective adjunctive therapy in REPs.
Assuntos
Terapia com Luz de Baixa Intensidade , Endodontia Regenerativa , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ácido Edético/farmacologia , Dentina/metabolismo , Polpa Dentária/metabolismo , Células-Tronco/metabolismoRESUMO
There are limited data on comparison of pulsed and continuous wave in photobiomodulation therapy (PBM). This study aimed to investigate the effect of PBM with 980 nm laser in pulsed and continuous wave on the proliferation and migration of human gingival fibroblasts (HGF) cells. Cultured HGF were divided into three main groups: (1) irradiated in pulsed mode (frequencies of 50 and 25 KHz; energy densities of 3 and 5 J/cm2 ), (2) irradiated in continuous mode (energy densities of 3.2 and 5.2 J/cm2 ), and (3) no irradiation as control group. HGF proliferation rate was measured by MTT assay at 24, 48, and 72 h post irradiation. In addition, HGF migration rate was measured by scratch test at 24 h post PBM. At 24 h, the group received continuous irradiation at 5.2 J/cm2 showed significantly higher proliferation compared with the control group (p = 0.012). At 48 and 72 h, the groups received continuous, and 50 Hz pulsed irradiation at energy densities of 5.2 and 5 J/cm2 respectively, had significantly higher HGF proliferation rates compared to the control (p < 0.05). Only the continuous irradiations were effective in significant increase of the cell migration. In conclusion, continuous PBM at energy density of 5.2 J/cm2 showed promising effect on HGF proliferation and migration.
Assuntos
Terapia com Luz de Baixa Intensidade , Humanos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular , Lasers , Fibroblastos/efeitos da radiaçãoRESUMO
The purpose of this study was to evaluate the effects of 635 nm diode laser with different powers on undifferentiated mesenchymal stem cells obtained from buccal fat pad. Human buccal fat stem cells were cultured in DMEM containing 10% FBS, penicillin, and streptomycin under 5% CO2 and 95% humidity. Cells were cultured in 96-well plate and 24 h later, laser irradiation with 635 nm diode laser was performed in four groups of 200, 300, 400, and 500 mW powers in addition to the control group with the same energy density of 4 J/cm2. MTT and flow cytometry assay was performed to evaluate cell proliferation and viability on 2 and 4 days after irradiation. Alizarin red assay and real-time PCR (OPN, OCN, ALP, and RUNX-2 genes) was performed to evaluate osteogenic differentiation. According to the MTT assay, none of the mentioned powers of 635 nm diode laser had significant effect on cell proliferation. Cells irradiated with power of 400 mW and 500 mW significantly showed a greater number of necrotic cells compared to the control group in Day 4. Cells irradiated with 300 mW power significantly exhibited a greater amount of nodule formation compared to all groups. Results of this study indicated that 635 nm diode laser with energy density of 4 J/cm2 has a positive effect inducing osteogenic differentiation when applying with a power of 300 mW in buccal fat pad mesenchymal stem cells.
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OBJECTIVE: Complications arising from diabetes can result in stem cell dysfunction, impairing their ability to undergo differentiation into various cellular lineages. The present study evaluated the effect of histone deacetylase inhibitors, Valproic acid and Trichostatin A, on the odontogenic differentiation potential of dental pulp stem cells under hyperglycemic conditions. METHODS: Streptozotocin (STZ) induced diabetes mellitus in 12 male Wistar rats. Dental parameters were examined using micro-computed tomography. The odontogenic potential of human pulp stem cells exposed to 30 mM glucose was assessed through alkaline phosphatase assays, examination of gene expression for dentin matrix protein 1 and dentin sialoprotein using real-time PCR, and alizarin red staining for calcium deposition. RESULTS: Along with reduced dentin thickness and root length in diabetic rats, the results revealed a significant increase in histone deacetylase 3 and 2 gene expressions in isolated diabetic pulp tissues compared to the control groups. The gene expression of odontogenic-related markers and alkaline phosphatase activity in human cultured pulp stem cells under hyperglycemic conditions significantly decreased. Adding Valproic acid and Trichostatin A restored the odontogenic differentiation markers, including calcium deposition, gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and alkaline phosphatase activity. CONCLUSION: The data suggests that hyperglycemic conditions negatively impact the odontogenic potential of pulp mesenchymal stem cells. However, histone deacetylase inhibitors improve the impaired odontogenic differentiation capacity. This study implies that histone deacetylases may represent a potential therapeutic target for enhancing the regenerative mineralization of pulp cells in diabetic patients.
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The anthracycline doxorubicin is a widely used effective anti-cancer drug. However, its application and dosage are severely limited due to its cardiotoxicity. The exact mechanisms of doxorubicin-induced cardiotoxic side effects remain poorly understood. Even less is known about the impact of doxorubicin treatment on vascular damage. We found that low doses of doxorubicin induced a senescent response in human primary vascular smooth muscle cells (VSMC). We observed that expression of urokinase receptor (uPAR) was upregulated in response to doxorubicin. Furthermore, the level of uPAR expression played a decisive role in developing doxorubicin-induced senescence. uPAR silencing in human VSMC by means of RNA interference as well as uPAR knockout in mouse VSMC resulted in abrogation of doxorubicin-induced cellular senescence. On the contrary, uPAR overexpression promoted VSMC senescence. We further found that proteasomal degradation of telomeric repeat binding factor 2 (TRF2) mediates doxorubicin-induced VSMC senescence. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in VSMC and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Therefore, VSMC senescence induced by low doses of doxorubicin may contribute to vascular damage upon doxorubicin treatment. uPAR-mediated TRF2 ubiquitination and proteasomal degradation are further identified as a molecular mechanism underlying this process.
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Senescência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Doxorrubicina/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas Recombinantes de Fusão/fisiologia , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína 2 de Ligação a Repetições Teloméricas/genética , Transfecção , Ubiquitinação/efeitos dos fármacos , Artérias Umbilicais/citologiaRESUMO
This study aimed to synthesize and characterize a novel dental pulp capping cement containing bioactive glass (BG)/zinc oxide modified with an organic resin. BG (45S5) with or without ZnO (Zn) and hemaphosphate (HP) combined with a liquid consisting of polyacrylic and itaconic acids (AA) were synthesized and the structural, physical, and mechanical properties were assessed. Hydroxyapatite formation was evaluated by immersion in simulated body fluid. Biological analysis including methyl thiazolyl tetrazolium assay, alizarin red staining, alkaline phosphatase (ALP) activity, and gene expression of odontogenic markers were performed to evaluate the cytotoxic effect and biomineralization potential of the cements on human dental pulp stem cells (hDPSCs). A commercial mineral trioxide aggregate (MTA) served as control. The highest compressive strength value and the shortest setting time were belonged to the BG + HP + AA and BG + AA groups, respectively. The shear bond strength to dentin was the highest for the BG + HP + AA cement. Scanning electron microscope showed only scarce deposits of calcium phosphate formation on the surface of the synthesized cements. BG + HP + AA and BG + HP + Zn + AA groups had significantly lower cytotoxicity than MTA. The mineralization potential of hDPSCs after stimulation by the novel cements increased. Quantitative reverse-transcription-polymerase chain reaction showed higher odontogenic marker expression in hDPSCs exposed to the BG + HP + Zn + AA cement compared to other synthesized cements, although it was higher in MTA group. Based on the obtained results, the novel synthesized cements can be used as appropriate capping agents in the treatment of dental pulp.
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Agentes de Capeamento da Polpa Dentária e Pulpectomia , Óxido de Zinco , Humanos , Vidro/química , Microscopia , Cimentos Dentários , Silicatos , Compostos de Cálcio , Óxidos , Combinação de MedicamentosRESUMO
Introduction. Antimicrobial sonodynamic therapy (aSDT) is an approach that uses ultrasound waves (UWs) and a sonosensitizer to generate reactive oxygen species (ROS) to damage microbial cells in biofilms. Using nano-carriers, such as exosomes (Exos), to deliver the sonosensitizer can potentially enhance the effectiveness of aSDT.Hypothesis/Gap Statement. aSDT can downregulate the expression of gelE and sprE genes, increasing the production of endogenous ROS and degradation of pre-formed Enterococcus faecalis biofilms.Aim. This study investigated the anti-biofilm effect of aSDT-based periodontal ligament stem cell-derived exosome-loaded kojic acid (KA@PDL-Exo) on pre-formed E. faecalis biofilms in root canals.Methodology. Following the isolation and characterization of PDL-Exo, KA@PDL-Exo was prepared and confirmed. The minimal biofilm inhibitory concentration (MBIC) of KA, PDL-Exo, KA@PDL-Exo and sodium hypochlorite (NaOCl) was determined, and their anti-biofilm effects were assessed with and without UWs. The binding affinity of KA with GelE and SprE proteins was evaluated using in silico molecular docking. Additionally, the study measured the generation of endogenous ROS and evaluated changes in the gene expression levels of gelE and sprE.Results. The results revealed a dose-dependent decrease in the viability of E. faecalis cells within biofilms. KA@PDL-Exo was the most effective, with an MBIC of 62.5 µg ml-1, while NaOCl, KA and PDL-Exo had MBIC values of 125, 250 and 500 µg ml-1, respectively. The use of KA@PDL-Exo-mediated aSDT resulted in a significant reduction of the E. faecalis biofilm (3.22±0.36 log10 c.f.u. ml-1; P<0.05). The molecular docking analysis revealed docking scores of -5.3 and -5.2 kcal mol-1 for GelE-KA an SprE-KA, respectively. The findings observed the most significant reduction in gene expression of gelE and sprE in the KA@PDL-Exo group, with a decrease of 7.9- and 9.3-fold, respectively, compared to the control group (P<0.05).Conclusion. The KA@PDL-Exo-mediated aSDT was able to significantly reduce the E. faecalis load in pre-formed biofilms, decrease the expression of gelE and srpE mRNA, and increase the generation of endogenous ROS. These findings imply that KA@PDL-Exo-mediated aSDT could be a promising anti-biofilm strategy that requires additional in vitro and in vivo investigations.
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Anti-Infecciosos , Exossomos , Enterococcus faecalis , Simulação de Acoplamento Molecular , Ligamento Periodontal , Espécies Reativas de Oxigênio , Células-Tronco , BiofilmesRESUMO
This study investigated the effect of photobiomodulation (PBM) with 980 nm diode laser as monotherapy and in combination with compressive and tensile orthodontic forces on expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), sclerostin (SOST) and periostin (POSTN), by human periodontal ligament cells. Isolated cells were cultured and subjected to either tensile (10% elongation) or compressive forces (25 g cm-2 ) for 24 and 48 h. Subsequently, the cells received PBM (100 mW power, 3 or 6 J cm-2 energy density) immediately after load cycle. RT-PCR was applied to assess the genes expression. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test (P ≤ 0.05). We found that PBM in combination with orthodontic forces led to upregulation of bone resorption genes (RANKL and SOST) at the pressure side and their downregulation at the tension side. The expression of osteogenic genes (OPG and POSTN) increased at the tension side and decreased at the pressure side. PBM alone did not affect gene expression. In conclusion, these findings suggest that this PBM protocol may be effective in enhancement of the gene expression in favor of bone remodeling acceleration that should be confirmed in future animal and human studies.
Assuntos
Terapia com Luz de Baixa Intensidade , Ligamento Periodontal , Animais , Humanos , Ligamento Periodontal/metabolismo , Lasers Semicondutores , Remodelação Óssea , Expressão Gênica , Células CultivadasRESUMO
OBJECTIVE: The present in vitro study aims to investigate the potential use of epigenetic inhibitors as treatment modalities in tongue squamous cell carcinoma. DESIGN: The human tongue squamous cell carcinoma cell line (CAL-27) was cultured and exposed to varying concentrations of 5-Azacitidine (5-Aza) or Trichostatin A (TSA) in the culture medium. The cell apoptosis was evaluated using Annexin V/PI by flow cytometry. To evaluate DNA damage response, γH2AX foci analysis was performed using immunofluorescence. Single cell gel electrophoresis (SCGE) was applied to measure DNA strand breaks. Gene expression was assessed by quantitative real-time PCR. RESULTS: The results showed that 5-Aza and TSA had apoptotic effects on the SCC cell line at concentrations of 50-200 µM and 0.5-5 µM, respectively. Immunofluorescence analysis showed increased expression of γH2AX, the marker of DNA damage response after treatment of 5-Aza and TSA that was associated with increased DNA strand breaks. The expressions of urokinase plasminogen activator, its receptor and matrix metalloproteinase-2, were significantly reduced in TSA- and 5-Aza-treated cells. CONCLUSIONS: Our results showed that 5-Aza and TSA increase apoptotic and DNA damage response in squamous cell carcinoma cell line while reducing the expression of tumor invasion genes that further indicating the potential therapeutic value of two epigenetic modifiers in squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas , Neoplasias da Língua , Azacitidina/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Dano ao DNA , Decitabina , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos , Metaloproteinase 2 da Matriz , Língua , Neoplasias da Língua/tratamento farmacológico , Neoplasias da Língua/genéticaRESUMO
Lead (Pb) is ubiquitous in environment that accumulates in teeth and calcified tissues from where it releases gradually with aging and adversely affects dental health. This study aimed to determine the effect of Pb exposure on odontogenic differentiation potential of isolated human dental pulp stem cells and investigate the possible underlying epigenetic factors. In the absence of Pb exposure, stem cells displayed significant odontogenic markers including elevated Alkaline Phosphatase (ALP) activity, Alizarin red staining intensity, and increased expression of odontogenic DMP1 and DSPP genes. Exposure to 60 µM Pb resulted in reduced ALP activity and calcium deposition. Also, diminished expression of RUNX2, DMP1, and DSPP, as well as Wnt signaling mediators including WNT1, and ß-catenin were detected. The expression of Wnt signaling related microRNAs, miRNA-139-5p and miRNA-142-3p, on the other hand, were shown to have a significant increase. We concluded that Pb could adversely affect the odontogenic differentiation potential of dental pulp stem cell. The underlying mechanism might related to Pb-induced epigenetic dysregulation of WNT1/ß-catenin pathway-related miRNAs leading to down-regulation of Wnt/ß-catenin related odontogenic genes and eventually impaired odontogenic differentiation process.