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1.
Braz. j. biol ; 84: e251970, 2024. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1345559

RESUMO

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.


Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.


Assuntos
Animais , Osteogênese , Fosfatase Alcalina/metabolismo , Rana catesbeiana , Osso e Ossos/metabolismo , Cinética
2.
Methods Mol Biol ; 2582: 281-291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370357

RESUMO

Dental pulp cells (DPCs) differentiate into odontoblasts. To observe odontoblastic differentiation, the detection of dentinogenesis-specific molecules such as dentin sialophosphoprotein (DSPP) and the measurement of alkaline phosphatase (ALP) activity are reliable approaches. CCN family member 2 (CCN2) has been proposed as a marker for dentinogenesis. Our recent study revealed that the expression levels of Ccn4, Ccn5, and Ccn6 were changed in accordance with odontoblastic differentiation. Therefore, Ccn4, Ccn5, and Ccn6, as well as Ccn2, could serve as a comprehensive set of markers for dentinogenesis. Here, we describe a method of measuring the Ccns expression levels in differentiating rat DPCs.


Assuntos
Fosfatase Alcalina , Odontoblastos , Ratos , Animais , Fosfatase Alcalina/metabolismo , Odontoblastos/metabolismo , Diferenciação Celular/genética , Expressão Gênica , Polpa Dentária/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células Cultivadas , Fosfoproteínas/metabolismo
3.
Dev Comp Immunol ; 138: 104494, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35940383

RESUMO

This study evaluated the epidermis mucosal capacity of goldfish (Carassius auratus) during different stages of reproductive development in both females and males. In this regard, the activity of mucolytic immune enzymes, i.e., lysozyme, complement and peroxidase, as well as the activity of alkaline phosphatase (ALP) were evaluated. There were five stages for females i.e., immature (f1), cortical alveoli (f2), early and late-vitellogenesis (vtg) (f3 and f4) and ripe (f5); as well as two stages for males spermatogenesis (m1) and spermiation (m2). Some stages were also examined for the mucosal antimicrobial activity against specific pathogens. The results showed that the mucosal lysozyme activity increased significantly during vitellogenesis (P < 0.05), but no lysozyme activity was detected in plasma. On the contrary, the complement activity was only observed in female plasma, and it was significantly higher at f3 compared to the other developmental stages. Both the plasma and mucosal ALP and peroxidase activities showed a significant increase by female reproductive development with the highest amounts at f4. Contrary to the female, no significant changes were observed in plasma and mucosal immune agents and biochemistry of the male. The f5-staged goldfish showed the highest antimicrobial activities against Gram-positive bacteria, i.e., Streptococcus faecium, Staphylococcus aureus and Micrococcus luteus (P < 0.05). Our results also represented the up-regulation of lysozyme (c-lys) gene expression by effects of female maturational development in ovary, liver and skin, while male goldfish showed no significant changes in c-lys expression. Moreover, there were positive correlations between c-lys expression, mucosal lysozyme activity and calcium levels in females (P < 0.01). Overall, our findings revealed that vtg process improves mucosal innate immunity that leads to activate antimicrobial components at spawning season.


Assuntos
Anti-Infecciosos , Carpa Dourada , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Anti-Infecciosos/metabolismo , Cálcio/metabolismo , Epitélio , Expectorantes/metabolismo , Feminino , Carpa Dourada/genética , Masculino , Peroxidases/genética , Peroxidases/metabolismo , Regulação para Cima
4.
J Biomed Mater Res B Appl Biomater ; 111(1): 151-160, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35950464

RESUMO

The development of nanoscale biomaterials associated with polymers has been growing over the years, due to their important structural characteristics for applications in biological systems. The present study aimed to produce and test polymeric scaffolds composed of polylactic acid (PLA) fibers associated with a 58S bioglass doped with therapeutic ions for use in tissue engineering. Three 58S Bioglass was obtained by the sol-gel route, pure and doped with 5% strontium and cobalt ions. Solutions of 7% PLA was used as control and added the three different bioglass, 4% of 58S bioglass (PLA-BG), 4% bioglass-doped strontium (PLA-BGSr) and 4% bioglass-doped cobalt (PLA-BGCo). Scaffolds were produced through electrospinning process, and was characterized chemical and morphologically. The in vitro tests were performed using mesenchymal cells cultures from femurs of nine rats, grown in osteogenic supplemented total culture medium. After osteoblastic differentiation induction cell viability, alkaline phosphatase activity, total protein content quantification, and visualization of mineralization nodule tests were performed. Analysis of normal distribution used the Shapiro-Wilk test (nanofibers diameter and biological assay). Data were compared using the Kruskal-Wallis nonparametric test (p = 0.05). The bioglasses produced proved to be free of nitrate, chlorinated and nano-sized, with effective incorporation of therapeutic ions in their structure. All materials showed cell viability (>70%), total protein production, and alkaline phosphatase activity. It was possible to develop polylactic acid scaffolds associated with 58S bioglass doped with therapeutic ions without cytotoxicity. Scaffolds characteristics appear to sustain its application in bone tissue engineering.


Assuntos
Estrôncio , Engenharia Tecidual , Ratos , Animais , Estrôncio/farmacologia , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Cobalto/farmacologia , Poliésteres/química , Osteogênese , Íons
5.
Int J Mol Sci ; 23(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36362051

RESUMO

To develop new alkaline phosphatase inhibitors (ALP), a series of pyrazolo-oxothiazolidine derivatives were synthesized and biologically assessed, and the results showed that all of the synthesized compounds significantly inhibited ALP. Specifically, compound 7g displayed the strongest inhibitory activity (IC50 = 0.045 ± 0.004 µM), which is 116-fold more active than monopotassium phosphate (IC50 = 5.242 ± 0.472 µM) as a standard reference. The most potent compound among the series (7g) was checked for its mode of binding with the enzyme and shown as non-competitively binding with the target enzyme. The antioxidant activity of these compounds was examined to investigate the radical scavenging effect. Moreover, the MTT assay method was performed to evaluate their toxic effects on the viability of MG-63 human osteosarcoma cells, and all compounds have no toxic effect on the cells at 4 µM. Computational research was also conducted to examine the binding affinity of the ligands with alkaline phosphatase, and the results revealed that all compounds showed good binding energy values within the active site of the target. Therefore, these novel pyrazolo-oxothiazolidine derivatives might be employed as promising pharmacophores for potent and selective alkaline phosphatase inhibitors.


Assuntos
Fosfatase Alcalina , Inibidores Enzimáticos , Humanos , Simulação de Acoplamento Molecular , Fosfatase Alcalina/metabolismo , Cinética , Relação Estrutura-Atividade , Inibidores Enzimáticos/farmacologia , Estrutura Molecular
6.
Sci Rep ; 12(1): 19852, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400944

RESUMO

Pseudoxanthoma elasticum (PXE) is a multisystem, genetic, ectopic mineralization disorder with no effective treatment. Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). This study evaluated the anticalcification effects of DS-1211, an orally administered, potent, and highly selective small molecule TNAP inhibitor, in mouse models of PXE. Calcium content in vibrissae was measured in KK/HlJ and ABCC6-/- mice after DS-1211 administration for 13-14 weeks. Pharmacokinetic and pharmacodynamic effects of DS-1211 were evaluated, including plasma alkaline phosphatase (ALP) activity and biomarker changes in PPi and pyridoxal-phosphate (PLP). Anticalcification effects of DS-1211 through TNAP inhibition were further evaluated in ABCC6-/- mice with genetically reduced TNAP activity, ABCC6-/-/TNAP+/+ and ABCC6-/-/TNAP+/-. In KK/HlJ and ABCC6-/- mouse models, DS-1211 inhibited plasma ALP activity in a dose-dependent manner and prevented progression of ectopic calcification compared with vehicle-treated mice. Plasma PPi and PLP increased dose-dependently with DS-1211 in ABCC6-/- mice. Mice with ABCC6-/-/TNAP+/- phenotype had significantly less calcification and higher plasma PPi and PLP than ABCC6-/-/TNAP+/+ mice. TNAP plays an active role in pathomechanistic pathways of dysregulated calcification, demonstrated by reduced ectopic calcification in mice with lower TNAP activity. DS-1211 may be a potential therapeutic drug for PXE.


Assuntos
Calcinose , Pseudoxantoma Elástico , Camundongos , Animais , Pseudoxantoma Elástico/tratamento farmacológico , Pseudoxantoma Elástico/genética , Fosfatase Alcalina/metabolismo , Modelos Animais de Doenças , Fenótipo , Calcinose/tratamento farmacológico , Calcinose/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética
7.
Cells ; 11(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36359794

RESUMO

Regenerative endodontic treatment based on tissue engineering has recently gained interest in contemporary restorative dentistry. However, low survival rates and poor potential differentiation of stem cells could undermine the success rate of pulp regenerative therapy. Human gingival fibroblast-conditioned medium (hGF-CM) has been considered a potential therapy for tissue regeneration due to its stability in maintaining multiple factors essential for tissue regeneration compared to live cell transplantation. This study aimed to investigate the potency of hGF-CM on stem cells from human dental pulp (DPSC) in pulp regeneration. A series of experiments confirmed that hGF-CM contributes to a significant increase in proliferation, migration capability, and cell viability of DPSC after H2O2 exposure. Moreover, it has been proved to facilitate the odontogenic differentiation of DPSC via qRT-PCR, ALP (alkaline phosphatase), and ARS (Alizarin Red S) staining. It has been discovered that such highly upregulated odontogenesis is related to certain types of ECM proteins (collagen and laminin) from hGF-CM via proteomics. In addition, it is found that the ERK pathway is a key mechanism via inhibition assay based on RNA-seq result. These findings demonstrate that hGF-CM could be beneficial biomolecules for pulp regeneration.


Assuntos
Meios de Cultivo Condicionados , Polpa Dentária , Peróxido de Hidrogênio , Engenharia Tecidual , Humanos , Fosfatase Alcalina/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Fibroblastos/metabolismo , Regeneração , Gengiva/citologia , Gengiva/metabolismo , Engenharia Tecidual/métodos
8.
Int J Mol Sci ; 23(21)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36361965

RESUMO

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.


Assuntos
Apigenina , Calcificação Fisiológica , Humanos , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Anexina A6/efeitos dos fármacos , Anexina A6/metabolismo , Apigenina/farmacologia , Apigenina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
9.
Oxid Med Cell Longev ; 2022: 8385456, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36193077

RESUMO

The study aimed to explore the feasibility of a nanodrug delivery system to treat open fractures with bone defects. We developed a cefazolin (Cef)/bone morphogenetic protein 2 (BMP-2)@mesoporous silica nanoparticle (MSN) delivery system; meanwhile, Cef/MBP-2@ poly(lactic-co-glycolic acid) (PLGA) was also developed as control. For the purpose of determining the osteogenic and anti-inflammatory actions of the nanodelivery system, we cultured bone marrow mesenchymal stem cells (BMSCs) and constructed a bone defect mouse model to evaluate its clinical efficacy. After physicochemical property testing, we determined that MSN had good stability and did not easily accumulate or precipitate and it could effectively prolong the Cef's half-life by nearly eight times. In BMSCs, we found that compared with the PLGA delivery system, MSNs better penetrated into the bone tissue, thus effectively increasing BMSCs' proliferation and migration ability to facilitate bone defect repair. Furthermore, the MSN delivery system could improve BMSCs' mineralization indexes (alkaline phosphatase [ALP], osteocalcin [OCN], and collagen I [Col I]) to effectively improve its osteogenic ability. Moreover, the MSN delivery system could inhibit inflammation in bone defect mice, which was mainly reflected in its ability to reduce the release of IL-1ß and IL-4 and increase IL-10 levels; it could also effectively reduce apoptosis of CD4+ and CD8+ T cells, thus improving their immune function. Furthermore, the percentage of new bones, bone mineral density, trabecular volume, and trabecular numbers in the fracture region were improved in mice treated with MSN, which allowed better repair of bone defects. Hence, Cef/BMP-2@MSN may be feasible for open fractures with bone defects.


Assuntos
Fraturas Expostas , Nanopartículas , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/uso terapêutico , Linfócitos T CD8-Positivos/metabolismo , Cefazolina/farmacologia , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Camundongos , Nanopartículas/química , Osteocalcina , Osteogênese , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Dióxido de Silício/química
10.
Chem Commun (Camb) ; 58(91): 12720-12723, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314354

RESUMO

Herein, we develop a hierarchically mesoporous cerium metal-organic framework (Ce-HMMOF) nanozyme with enhanced ALP-mimicking activity for the naked-eye detection of phosphorylated biomarkers. The long-range ordered mesochannels (9.18 nm) throughout the Ce-HMMOF promote both the mass transfer and the accessibility of interior active sites, permitting the rapid and sensitive sensing of phosphorylated biomarkers through ALP-like biocatalysis. This work provides a new insight into the engineering of highly active nanozymes for disease-associated biomarker screening and diagnosis.


Assuntos
Cério , Estruturas Metalorgânicas , Fosfatase Alcalina/metabolismo , Estruturas Metalorgânicas/química , Cério/química , Biocatálise , Biomarcadores
11.
Drug Des Devel Ther ; 16: 3327-3342, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36199629

RESUMO

Aim: Liver regulates metabolism of biomolecules and injury of liver causes distortion of metabolic functions. This injury may be oxidative or inflammatory induced by numerous factors including alcohol, pathogens and xenobiotics. This scientific study was planned to investigate the anti-inflammatory and anti-oxidant potential of p-coumaric acid (p-CA) on Lipopolysaccharide/D-Galactosamine (LPS/D-GalN) induced liver injury. Methods: DPPH analysis, reducing power assay and HPLC analysis were performed during in-vitro studies of p-CA. Similarly, in-vivo experiments were performed using Wistar Albino rats. Normal control and intoxicated group received (5mL/kg normal saline p.o), standard treatment groups received ascorbic acid (100mg/kg p.o) and silymarin (25mg/kg p.o), while p-CA treatment groups received (100mg/kg p.o) for 28-days. After completion of 28-days, LPS/D-GalN injection (300 mg D-GalN/kg and 10 µg LPS/kg i.p.) was given at 6th, 12th and 24-hours to all groups except normal control group. Animals were sacrificed; serum and liver samples were harvested and subjected to biochemical and histological examinations, respectively. Results: The results revealed that p-CA possess strong antioxidant activity. Increased levels of leukocyte infiltration (TLC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), lipid panel (eg TG, TC, LDL-C, VLDL-C), whereas decreased HDL-C levels noticed in LPS/D-GalN groups as compared to normal control groups. Pro-Inflammatory markers (eg TNF-α, IL-6, IL-1ß) and lipid peroxidation marker, eg malondialdehyde (MDA) increased while superoxide dismutase (SOD) and reduced glutathione (GSH) levels were decreased significantly in groups treated with LPS/D-GalN. ANOVA with Bonferroni post hoc analysis was used for statistical analysis of. H&E staining was done to assess architectural abnormalities among liver cells. Conclusion: In conclusion, p-CA could ameliorate LPS/D-GalN induced hepatic injury via regulation of immune responses, liver function enzymes, lipid profile, oxidative stress and pro-inflammatory markers.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Silimarina , Alanina Transaminase/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Aspartato Aminotransferases/metabolismo , Bilirrubina , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , LDL-Colesterol , Ácidos Cumáricos , Galactosamina/farmacologia , Glutationa/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Fígado , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Solução Salina/farmacologia , Silimarina/farmacologia , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Shanghai Kou Qiang Yi Xue ; 31(3): 237-242, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-36204949

RESUMO

PURPOSE: To investigate the effects of microRNA-31-5p (miR-31-5p) on the signal pathway of hypoxia inducible factor-1α (HIF-1α)/Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3) and the expression of osteoblast-related factors of dental pulp stem cells(DPSCs). METHODS: Human dental pulp stem cells (DPSCs) were cultured in vitro and divided into the control group (no transfection), mimic NC group (transfected with negative control-miR-31-5p), miR-31-5p mimic group (transfected with hsa-miR-31-5p mimic), siRNA NC group (transfected with nonsense siRNA) and miR-31-5p siRNA group (transfected with miR-31-5p siRNA).The expressions of miR-31-5p, HIF-1α, BNIP3, alkaline phosphatase(ALP) and Runt-related transcription factor-2(Runx2) mRNA in DPSCs were detected by real-time fluorescence quantitative PCR; the proliferation of DPSCs was detected by MTT; ALP activity of DPSCs was detected by ALP activity test kit; and the protein expressions of HIF-1α, BNIP3 and Runx2 in DPSCs were detected by Western blot. Statistical analysis was carried out with SPSS 24.0 software package. RESULTS: Compared with the control group and mimic NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p mimic group were significantly lower (P<0.05), ALP staining decreased significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly higher (P<0.05). Compared with the control group and siRNA NC group, the A value, ALP mRNA expression level and activity, Runx2 mRNA and protein expression levels of DPSCs in miR-31-5p siRNA group were significantly higher (P<0.05), ALP staining enhanced significantly, and the expression levels of miR-31-5p mRNA, HIF-1α, BNIP3 mRNA and HIF-1α, BNIP3, Beclin1 protein were significantly lower(P<0.05). CONCLUSIONS: MiR-31-5p may inhibit the expression of osteoblast-related factors of DPSCs, and activating HIF-1α/BNIP3 signaling pathway.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , MicroRNAs , Fosfatase Alcalina/metabolismo , Proteína Beclina-1/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Polpa Dentária/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo
13.
Shanghai Kou Qiang Yi Xue ; 31(3): 243-247, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-36204950

RESUMO

PURPOSE: To investigate the effects of inflammatory microenvironment on proliferation and osteogenic differentiation of periodontal ligament cells(PDLCs) in vitro. METHODS: Human PDLCs were isolated and characterized. MTT was used to investigate the proliferation rate of PDLCs under different concentration of lipopolysaccharide(LPS). The PDLCs' osteogenic differentiation was investigated using real-time PCR and Western blot. The date were statistically analyzed with SPSS 13.0 software package. RESULTS: Treatment with 0.1 µg/mL LPS increased proliferation of PDLCs and enhanced the expression of osteogenic gene and protein. The proliferation of PDLCs and expression of alkaline phosphatase(ALP), RUNX2, Collagen-I, BMP2 were significantly decreased by 10 µg/mL LPS. CONCLUSIONS: The inflammatory microenvironment (10 µg/mL LPS) inhibits the proliferation and osteogenic differentiation of human PDLCs.


Assuntos
Osteogênese , Ligamento Periodontal , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Inflamação , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Osteogênese/genética , Ligamento Periodontal/metabolismo
14.
PLoS One ; 17(10): e0268592, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36206263

RESUMO

Fetuin-A is a liver derived plasma protein showing highest serum concentrations in utero, preterm infants, and neonates. Fetuin-A is also present in cerebrospinal fluid (CSF). The origin of CSF fetuin-A, blood-derived via the blood-CSF barrier or synthesized intrathecally, is presently unclear. Fetuin-A prevents ectopic calcification by stabilizing calcium and phosphate as colloidal calciprotein particles mediating their transport and clearance. Thus, fetuin-A plays a suppressive role in inflammation. Fetuin-A is a negative acute-phase protein under investigation as a biomarker for multiple sclerosis (MS). Here we studied the association of pediatric inflammatory CNS diseases with fetuin-A glycosylation and phosphorylation. Paired blood and CSF samples from 66 children were included in the study. Concentration measurements were performed using a commercial human fetuin-A/AHSG ELISA. Of 60 pairs, 23 pairs were analyzed by SDS-PAGE following glycosidase digestion with PNGase-F and Sialidase-AU. Phosphorylation was analyzed in 43 pairs by Phos-TagTM acrylamide electrophoresis following alkaline phosphatase digestion. Mean serum and CSF fetuin-A levels were 0.30 ± 0.06 mg/ml and 0.644 ± 0.55 µg/ml, respectively. This study showed that serum fetuin-A levels decreased in inflammation corroborating its role as a negative acute-phase protein. Blood-CSF barrier disruption was associated with elevated fetuin-A in CSF. A strong positive correlation was found between the CSF fetuin-A/serum fetuin-A quotient and the CSF albumin/serum albumin quotient, suggesting predominantly transport across the blood-CSF barrier rather than intrathecal fetuin-A synthesis. Sialidase digestion showed increased asialofetuin-A levels in serum and CSF samples from children with neuroinflammatory diseases. Desialylation enhanced hepatic fetuin-A clearance via the asialoglycoprotein receptor thus rapidly reducing serum levels during inflammation. Phosphorylation of fetuin-A was more abundant in serum samples than in CSF, suggesting that phosphorylation may regulate fetuin-A influx into the CNS. These results may help establish Fetuin-A as a potential biomarker for neuroinflammatory diseases.


Assuntos
Cálcio , alfa-2-Glicoproteína-HS , Acrilamidas/metabolismo , Proteínas de Fase Aguda/metabolismo , Fosfatase Alcalina/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Biomarcadores , Cálcio/metabolismo , Doenças do Sistema Nervoso Central , Criança , Glicosilação , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Inflamação/metabolismo , Fígado/metabolismo , Neuraminidase/metabolismo , Fosfatos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Albumina Sérica/metabolismo , alfa-2-Glicoproteína-HS/metabolismo , alfa-Fetoproteínas/metabolismo
15.
Stem Cell Res Ther ; 13(1): 495, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195958

RESUMO

BACKGROUND: The repair of cranio-maxillofacial bone defects remains a formidable clinical challenge. The Ets variant 2 (ETV2) transcription factor, which belongs to the E26 transformation-specific (ETS) family, has been reported to play a key role in neovascularization. However, the role of ETV2 in the osteogenesis of human dental pulp stem cells (hDPSCs) remains unexplored. METHODS: Transgenic overexpression of ETV2 was achieved using a lentiviral vector, based on a Dox-inducible system. The effects of Dox-induced overexpression of ETV2 on the osteogenesis of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, RNA-sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of ETV2-induced osteogenesis. Additionally, the role of ETV2 overexpression in bone formation in vivo was validated by animal studies with a rat calvarial defect model and a nude mice model. RESULTS: Our results demonstrated that ETV2 overexpression significantly upregulated the mRNA and protein expression levels of osteogenic markers, markedly enhanced ALP activity, and promoted matrix mineralization of hDPSCs. Moreover, the results of RNA-Seq analysis and western blot showed that the ERK/MAPK and PI3K-Akt signaling pathways were activated upon transgenic overexpression of ETV2. The enhanced osteogenic differentiation of hDPSCs due to ETV2 overexpression was partially reversed by treatment with inhibitors of ERK/MAPK or PI3K-AKT signaling. Furthermore, the results of in vivo studies demonstrated that ETV2 overexpression improved bone healing in a rat calvarial defect model and increased ectopic bone formation in nude mice. CONCLUSIONS: Collectively, our results indicated that ETV2 overexpression exerted positive effects on the osteogenesis of hDPSCs, at least partially via the ERK/MAPK and PI3K/AKT signaling pathways.


Assuntos
Osteogênese , Fosfatidilinositol 3-Quinases , Fatores de Transcrição , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Polpa Dentária/metabolismo , Humanos , Camundongos , Camundongos Nus , Osteogênese/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
16.
Sci Rep ; 12(1): 17410, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36258024

RESUMO

The fish immune system is a topic or subject that offers a unique understanding of defensive system evolution in vertebrate heredity. While gut microbiota plays several roles in fish: well-being, promoting health and growth, resistance to bacterial invasion, regulation of energy absorption, and lipid metabolism. However, studies on fish gut microbiota face practical challenges due to the large number of fish varieties, fluctuating environmental conditions, and differences in feeding habits. This study was carried out to evaluate the impacts of supplemented three autochthonous strains, Bacillus sp. RCS1, Pantoea agglomerans RCS2, and Bacillus cereus RCS3 mixture diet on cobia fish (Rachycentron canadum). Also, chromatography, mass spectrometry and high throughput sequencing were combined to explore composition and metabolite profile of gut microbiota in juvenile cobia fed with supplemented diet. In the trial group, juvenile cobia received diets supplemented with 1 × 1012 CFU mL-1 autochthonous strains for ten weeks and a control diet without supplementation. Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without additives (control). Haematological indices, such as red blood cells, white blood cells, corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin, and mean corpuscular haemoglobin, were higher in the supplemented group. Similarly, digestive enzymes (trypsin, lipase, amylase, pepsin and cellulose, activities) activities were higher in supplemented diet with an indigenous isolates mixture. Serum biochemical parameters albumin, globulin, and total protein were significantly higher, while triglyceride, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and cholesterol showed no significant difference. On the other hand, glucose was significantly (P < 0.05) higher in the group without supplementation. On gene expression in the midgut, Immunoglobulin, Colony-stimulating factor receptor 1, major histocompatibility complex 1 were up-regulated by native isolates while T cell receptor beta, and Major histocompatibility complex 2 showed no significant difference. Gut bacterial composition was altered in fish receiving supplemented diet with autochthonous strains. Metabolomics also revealed that some metabolic pathways were considerably enriched in fish fed with supplemented diet; pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that differentially expressed metabolites were involved in galactose metabolism, tryptophan metabolism, carbohydrate digestion and absorption, purine metabolism, and ABC transporters. Functional analysis of bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites, nucleotide and its metabolites, amino acid and its metabolites, heterocyclic compounds, and tryptamines, cholines, pigments. The current investigation results showed that autochthonous strains mixture has significantly enhanced the growth, survival, and innate and adaptive immunities of juvenile cobia.


Assuntos
Microbioma Gastrointestinal , Perciformes , Animais , Alanina/metabolismo , Albuminas/metabolismo , Fosfatase Alcalina/metabolismo , Aminoácidos/metabolismo , Amilases/metabolismo , Ração Animal/análise , Aspartato Aminotransferases/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Celulose/metabolismo , Colesterol/metabolismo , Dieta , Peixes/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Lipase/metabolismo , Metaboloma , Nucleotídeos/metabolismo , Pepsina A/metabolismo , Perciformes/fisiologia , Purinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Fator Estimulador de Colônias/metabolismo , Triglicerídeos/metabolismo , Tripsina/metabolismo , Triptaminas , Triptofano/metabolismo
17.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232787

RESUMO

Human dental pulp stem cells (hDPSCs) are multipotent mesenchymal stem cells (MSCs) that are capable of self-renewal with multilineage differentiation potential. After being cryopreserved, hDPSCs were reported to maintain a high level of proliferation and multi-differentiation abilities. In order to optimize cryopreservation techniques, decrease storage requirements and lower contamination risks, the feasibility of new whole-tooth cryopreservation and its effects on hDPSCs were tested. The survival rates, morphology, proliferation rates, cell activity, surface antigens and differentiation abilities of hDPSCs isolated from fresh teeth were compared with those of one-month cryopreserved teeth in 5% and 10% DMSO. The data of the present study indicated that the new cryopreservation approach did not reduce the capabilities or stemness of hDPSCs, with the exception that it extended the first appearance time of hDPSCs in the teeth that were cryopreserved in 10% DMSO, and reduced their recovery rate. With the novel strategy of freezing, the hDPSCs still expressed the typical surface markers of MSCs and maintained excellent proliferation capacity. Three consecutive weeks of osteogenic and adipogenic induction also showed that the expression of the key genes in hDPSCs, including lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), type I collagen (COL I) and osteocalcin (OSC) was not affected, indicating that their differentiation abilities remained intact, which are crucial parameters for hDPSCs as cell-therapy candidates. These results demonstrated that the new cryopreservation method is low-cost and effective for the good preservation of hDPSCs without compromising cell performance, and can provide ideas and evidence for the future application of stem-cell therapies and the establishment of dental banks.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Lipase Lipoproteica , Fosfatase Alcalina/metabolismo , Antígenos de Superfície/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Criopreservação/métodos , Polpa Dentária/metabolismo , Dimetil Sulfóxido/metabolismo , Humanos , Lipase Lipoproteica/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Células-Tronco/metabolismo
18.
Int J Implant Dent ; 8(1): 39, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36184700

RESUMO

PURPOSE: To compare the release of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-I) and interleukin 1ß (IL-1ß) of plasma rich in growth factors (PRGF) and leucocyte platelet-rich fibrin (L-PRF) and to evaluate their biological implication in osteoblasts. METHODS: Blood from 3 healthy volunteers was processed into PRGF, immediate L-PRF (L-PRF 0') and L-PRF 30 min after collection (L-PRF-30') and a control group. Growth factors release were analyzed at 7 times by ELISA. Cell proliferation, collagen-I synthesis and alkaline phosphatase activity were assessed in primary cultures of human osteoblasts. RESULTS: A slower controlled release of IGF-I, VEGF and PDGF was observed in the PRGF group at day 14. A higher synthesis of type I collagen was also quantified in PRGF. L-PRF released significantly higher amounts of IL-1ß, that was almost absent in the PRGF. CONCLUSIONS: The addition of leukocytes dramatically increases the secretion of proinflammatory cytokines, which are likely to negatively influence the synthesis of type I collagen and alkaline phosphatase (ALP) by osteoblasts.


Assuntos
Fibrina Rica em Plaquetas , Fosfatase Alcalina/metabolismo , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Preparações de Ação Retardada/metabolismo , Fibrina/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-1beta/metabolismo , Leucócitos , Osteoblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Biochem Biophys Res Commun ; 632: 32-39, 2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36194917

RESUMO

MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteoblastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21-5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21-5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21-5p and Dec1 deficiencies on bone formation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and measurements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21-5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21-5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21-5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of-function transfection study with a miR-21-5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21-5p. These findings showed that the reduced osteogenic potential due to a miR-21-5p deficiency is achieved by enhanced Dec1 expression and that the miR-21-5p/Dec1 axis is involved in regulating osteoblast function.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , MicroRNAs , Osteoblastos , Osteogênese , Animais , Camundongos , Fosfatase Alcalina/metabolismo , Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Osteopontina/metabolismo , Cloreto de Tolônio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microtomografia por Raio-X , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo
20.
Oxid Med Cell Longev ; 2022: 6360133, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36275897

RESUMO

Objective: Glucocorticoid-induced osteonecrosis of the femoral head is one of the most common causes of nontraumatic osteonecrosis of the femoral head, but its exact pathogenesis remains unclear. The aim of this study was to investigate the role of SIRT6 in the maintenance of bone tissue morphology and structure, intravascular lipid metabolism, and its potential molecular mechanism in glucocorticoid-induced osteonecrosis of the femoral head. Methods: SIRT6 adenovirus was transfected into GIONFH in rats. The microstructure of rat bone was observed by micro-CT and histological staining, and the expression of bone formation-related proteins and angiogenesis-related factors was determined through western blot and immunohistochemistry. Alkaline phosphatase activity, alizarin red staining, and the expression levels of Runx2 and osteocalcin were used to evaluate the osteogenic potential. And in vitro tube formation assay and immunofluorescence were used to detect the ability of endothelial cell angiogenesis. Results: Dexamethasone significantly inhibited osteoblast differentiation, affected bone formation, and destroyed microvessel formation, increased the intracellular Fe2+ and ROS levels and induced the occurrence of ferroptosis. SIRT6 can inhibit ferroptosis and restore the ability of bone formation and angiogenesis. Conclusion: SIRT6 can inhibit the occurrence of ferroptosis, reduce the damage of vascular endothelium, and promote osteogenic differentiation, so as to prevent the occurrence of osteonecrosis of the femoral head.


Assuntos
Osteonecrose , Sirtuínas , Animais , Ratos , Fosfatase Alcalina/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dexametasona , Cabeça do Fêmur/metabolismo , Glucocorticoides/farmacologia , Osteocalcina/metabolismo , Osteogênese , Osteonecrose/induzido quimicamente , Osteonecrose/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/metabolismo
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