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1.
J Appl Oral Sci ; 28: e20190409, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32267378

RESUMEN

BACKGROUND: Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. OBJECTIVE: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. METHODOLOGY: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. RESULTS: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. CONCLUSION: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.


Asunto(s)
Proceso Alveolar/fisiopatología , Fuerza de la Mordida , Factor I del Crecimiento Similar a la Insulina/análisis , Osteoprotegerina/análisis , Ovariectomía , Ligando RANK/análisis , Fosfatasa Alcalina/sangre , Animales , Western Blotting , Ensayo de Immunospot Ligado a Enzimas , Estradiol/sangre , Femenino , Osteocalcina/sangre , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Microtomografía por Rayos X
2.
Int. j. morphol ; 38(2): 400-405, abr. 2020. graf
Artículo en Inglés | LILACS | ID: biblio-1056454

RESUMEN

Accumulating evidence from preclinical and clinical studies indicates prenatal exposure to stress or excess glucocorticoids can affect offspring brain. Glucocorticoid receptor (GR) is an important target of glucocorticoid. Therefore the aim of the present study was to investigate the expression of GR in prenatally stressed adult offspring and the relationship between GR expression and behavior in offspring. Pregnant rats received restraint stress during the last week of pregnancy. Hippocampal glucocorticoid receptor expression levels in the offspring were detected on postnatal 60 (P60).Cognition function was also detected. It shows significantly lower hippocampal GR expression was observed in female prenatally stressed offspring compared with their controls at P60. Corresponding to the expression of GR, female prenatally stressed offspring exhibited poorer spatial learning and memory abilities in the Barnes maze than control, This suggests that cognitive impairment in prenatally stressed rat offspring attribute lower hippocampal GR expression.


La evidencia acumulada de estudios preclínicos y clínicos indica que la exposición prenatal al estrés, o el exceso de glucocorticoides puede afectar el desarrollo cerebral de las crías. El receptor de glucocorticoides (RG) es un objetivo importante de los glucocorticoides. Por lo tanto, el objetivo del presente estudio fue investigar la expresión de RG en crías adultas estresadas durante el período prenatal y la relación entre la expresión de RG y el comportamiento de las crías. Las ratas preñadas recibieron niveles de estrés restringido, durante la última semana de embarazo. Se determinaron niveles de expresión del receptor de glucocorticoides del hipocampo y niveles de función cognitiva en las crías. En comparación con el grupo control se observó una expresión de RG en el hipocampo, significativamente menor en las crías estresadas prenatalmente, en comparación con los controles en P60. En referencia a la expresión de RG, las crías estresadas prenatalmente exhibieron habilidades de memoria y aprendizaje espacial menores, en el laberinto de Barnes que el grupo control. Esto sugiere que el deterioro cognitivo en crías de ratas estresadas prenatalmente muestran una menor expresión de RG en el hipocampo.


Asunto(s)
Animales , Femenino , Embarazo , Ratas , Efectos Tardíos de la Exposición Prenatal , Receptores de Glucocorticoides/metabolismo , Disfunción Cognitiva , Hipocampo/metabolismo , Estrés Fisiológico , Inmunohistoquímica , Western Blotting , Ratas Sprague-Dawley
3.
Braz Oral Res ; 34: e030, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32236319

RESUMEN

The abnormal increase in proliferation rate of human periodontal ligament stem cells (PDLSCs) is considered to be involved in the pathogenesis of periodontitis, a disease in which the IL-10-mediated anti-inflammatory pathway plays a critical role. This study aimed to investigate the involvement of microRNA-466l in periodontitis and to explore the possible interaction between IL-10 and microRNA-466l. PDLSCs were obtained from periodontitis-affected teeth and healthy control teeth. The expression of microRNA-466l and IL-10 mRNA was measured in PDLSCs using RT-qPCR. The proliferation ability of PDLSCs was analyzed using CCK-8 assays. Overexpression of microRNA-466l in a PDLSC cell line was established using two different types of PDLSCs, and the effect of microRNA-466l overexpression on IL-10 expression and cell proliferation were detected by western blot and CCK-8 assays, respectively. We found that expression levels of microRNA-466l and IL-10 mRNA were significantly lower (P < 0.05) in PDLSCs derived from periodontitis-affected teeth compared to those derived from healthy teeth. However, the cell proliferation ability was significantly higher in the PDLSCs derived from periodontitis-affected teeth. Meanwhile microRNA-466l overexpression decreased cell proliferation rates of both types of PDLSCs and upregulated IL-10 expression. Together, these data suggest that microRNA-466l can upregulate IL-10 and reduce the proliferation rate of PDLSCs.


Asunto(s)
Proliferación Celular/fisiología , Interleucina-10/metabolismo , Interleucina-10/uso terapéutico , MicroARNs/metabolismo , Periodontitis/terapia , Células Madre/metabolismo , Adulto , Western Blotting , Diferenciación Celular , Humanos , Periodontitis/genética , Periodontitis/metabolismo , Regulación hacia Arriba
4.
Zhonghua Zhong Liu Za Zhi ; 42(3): 197-202, 2020 Mar 23.
Artículo en Chino | MEDLINE | ID: mdl-32252197

RESUMEN

Objective: To investigate the effects of metastasis associated gene 1 (MTA1) expression on the proliferation and apoptosis of human esophageal cancer Eca109 cells. Methods: MTA1 siRNA was transfected into human esophageal cancer Eca109 cells, and the control group and blank group were set up. The expression of MTA1 in Eca109 cells with different treatment was detected by real-time fluorescent quantitative PCR (RT-PCR) and western blot. The proliferation of Eca109 cells was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The cloning formation ability of Eca109 cells was detected by plate cloning assay. The apoptosis of Eca109 cells were detected by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related proteins, including cleaved caspase-3 and total caspase-3 protein in Eca109 cells were detected by western blot. Results: After 48 hours of transfection, RT-PCR result showed that the relative expression levels of MTA1 mRNA in Eca109 cells in the blank group, control group, and siRNA group were 1.00±0.10, 0.98±0.09 and 0.21±0.03, respectively. The expression of MTA1 mRNA in siRNA group was significantly inhibited (P<0.05), while no significant difference of MTA1 mRNA expression between the blank group and the control group has been found (P>0.05). Western blot results were consistent with those of RT-PCR. MTT array results showed that, compared with the blank group and transfection group, the absorbance values of Eca109 cells in siRNA group were dramatically reduced at 48, 72, and 96 h (all P<0.05). There were no significant differences of absorbance values between the blank group and the control group at 48, 72, and 96 h (all P>0.05). The results of the plate colony formation test showed that the number of colony formation in the blank group and control group were 58.64±6.86 and 60.02±7.04, respectively, significantly higher than 18.10±3.16 in siRNA group (P<0.05), while there was no significant difference between the blank group and control group (P>0.05). Flow cytometry results showed that the apoptosis rates in the blank group and control group were (2.13±0.54)% and (2.27±0.61)%, respectively, significantly lower than (32.61±5.28)% in siRNA group (P<0.05), while there was no significant difference between blank group and control group (P>0.05). Western blot results showed that the expression of PCNA protein was down-regulated while cleaved caspase-3 protein expression was upregulated in siRNA group, compared to the control group and blank group (P<0.05). Conclusions: Inhibition of MTA1 expression can inhibit the proliferation of Eca109 cells and induce apoptosis. This process may be related to the down-regulation of PCNA protein expression and activation of caspase-3 protein expression.


Asunto(s)
Apoptosis , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/fisiología , Proteínas Represoras/fisiología , Transactivadores/metabolismo , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Neoplasias Esofágicas/metabolismo , Histona Desacetilasas/genética , Humanos , Antígeno Nuclear de Célula en Proliferación , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Transfección
5.
Einstein (Sao Paulo) ; 18: eAO4560, 2020.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-32321078

RESUMEN

OBJECTIVE: To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERß) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. METHODS: Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. RESULTS: Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. CONCLUSION: The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.


Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Antagonistas del Receptor de Estrógeno/farmacología , Fulvestrant/farmacología , Receptores Acoplados a Proteínas G/agonistas , Análisis de Varianza , Western Blotting , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/antagonistas & inhibidores , Femenino , Citometría de Flujo/métodos , Humanos , Células MCF-7 , Receptores Acoplados a Proteínas G/análisis , Reproducibilidad de los Resultados , Sirolimus/farmacología , Factores de Tiempo , Transfección/métodos
6.
Mutat Res ; 852: 503165, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32265046

RESUMEN

Human risk assessment of genotoxic chemicals is an important area of research. However, the specificity of in vitro mammalian genotoxicity assays is sometime low, as they yield to misleading positive results that are not observe in in vivo studies. Apoptosis can be a confounding factor in the interpretation of the results. Recently, a new strategy for genotoxicity screening, based on the combined analysis of phosphorylated histones H2AX (γH2AX) and H3 (pH3), was proposed to discriminate efficiently aneugenic from clastogenic compounds. However, γH2AX biomarker could also be induce by apoptosis. The aim of the present study was to investigate the specificity of this genotoxic biomarker. For this purpose, we analyzed 26 compounds inducing apoptosis by different mechanism of action, with the γH2AX assay in three human cell lines after 24 h treatment. Most of the tested chemicals were negative in the assay, whatever the cell line tested. The few compounds that generated positive data have also been report positive in other genotoxicity assays. The data presented here demonstrate that the γH2AX assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. Currently, no formal guidelines have been approve for the γH2AX assay for regular genotoxicity studies, but we suggest that this biomarker could be used as a new standard genotoxicity assay.


Asunto(s)
Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Western Blotting/métodos , Histonas/genética , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Mutágenos/toxicidad , Apoptosis/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Células Hep G2 , Histonas/metabolismo , Humanos , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Mutágenos/clasificación , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo
7.
Braz Oral Res ; 34: e015, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32130362

RESUMEN

We sought to compare the characteristics and clinical significance of neutrophil extracellular traps in gingival samples from patients with periodontitis and those with gingivitis. The clinical indexes of gingival samples from patients with periodontitis and gingivitis were measured; the expression of TNF-alpha and IL-8 was measured by real-time fluorescent quantitative PCR; and the expression of TLR-8 and MMP-9 was measured by western blotting assays. Chemotaxis, phagocytosis and phagocytic activity of neutrophils were measured. Compared with the healthy group, the expression of TNF-α and IL-8 in the periodontitis group and the gingivitis group increased significantly (p < 0.05), and TNF-α in the gingivitis group was significantly lower than that in the healthy group (p < 0.05). The expression of IL-8 in the periodontitis group was significantly higher than that in the periodontitis group (p < 0.05). Furthermore, the expression of TLR-8 and MMP-9 in the periodontitis group was different from that in the gingivitis group and the healthy group, and the expression of TLR-8 and MMP-9 in the gingivitis group was significantly different from that in the healthy group (p < 0.05). In addition, the neutrophil mobility index in healthy people was 3.02 ± 0.53, that in the periodontitis group was 2.21 ± 0.13, and that in the gingivitis group was 2.31 ± 0.12. In conclusion, the chemotaxis of neutrophils in gingival samples of patients with periodontitis and gingivitis was decreased, the phagocytotic ability and activity of neutrophils were reduced, and the release of the extracellular trap-releasing inducible factors TNF-alpha and IL-8 also declined.


Asunto(s)
Trampas Extracelulares , Gingivitis/patología , Neutrófilos/patología , Periodontitis/patología , Actinas/análisis , Adulto , Western Blotting , Estudios de Casos y Controles , Electroforesis en Gel de Agar , Femenino , Humanos , Interleucina-8/análisis , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Persona de Mediana Edad , Índice Periodontal , ARN/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Receptor Toll-Like 8/análisis , Factor de Necrosis Tumoral alfa/análisis , Adulto Joven
8.
Einstein (Sao Paulo) ; 18: eAO5022, 2020.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-32215468

RESUMEN

OBJECTIVE: To evaluate the effects of oxidative stress on insulin signaling in cardiac tissue of obese mice. METHODS: Thirty Swiss mice were equally divided (n=10) into three groups: Control Group, Obese Group, and Obese Group Treated with N-acetylcysteine. After obesity and insulin resistance were established, the obese mice were treated with N-acetylcysteine at a dose of 50mg/kg daily for 15 days via oral gavage. RESULTS: Higher blood glucose levels and nitrite and carbonyl contents, and lower protein levels of glutathione peroxidase and phosphorylated protein kinase B were observed in the obese group when compared with their respective control. On the other hand, treatment with N-acetylcysteine was effective in reducing blood glucose levels and nitrite and carbonyl contents, and significantly increased protein levels of glutathione peroxidase and phosphorylated protein kinase B compared to the Obese Group. CONCLUSION: Obesity and/or a high-lipid diet may result in oxidative stress and insulin resistance in the heart tissue of obese mice, and the use of N-acetylcysteine as a methodological and therapeutic strategy suggested there is a relation between them.


Asunto(s)
Acetilcisteína/farmacología , Dieta Alta en Grasa , Depuradores de Radicales Libres/farmacología , Resistencia a la Insulina/fisiología , Miocardio/metabolismo , Obesidad/metabolismo , Estrés Oxidativo/fisiología , Animales , Glucemia/análisis , Western Blotting , Peso Corporal , Fluoresceínas/análisis , Humanos , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica , Especies Reactivas de Oxígeno/análisis , Valores de Referencia , Espectrofotometría
9.
Rev Assoc Med Bras (1992) ; 66(1): 42-47, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32130380

RESUMEN

OBJECTIVE: ADAMTS4 is a member of the ADAMTS4 family, which secretes proteinases. The mechanism of tumor metastasis may be correlated to its promotion of angiogenesis. It was determined whether ADAMTS4 participates in colorectal cancer progression. METHODS: The expression in clinical samples and CRC cell lines was investigated. Using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RT-PCR, the expression of ADAMTS4 was determined in colorectal tumors of different cancer stages and anatomic sites, and in three cell lines of different aggressiveness. RESULTS: The overexpression of ADAMTS4 was observed in tissue samples by IHC, and this was mainly located in the cytoplasm, as detected by FISH. The qRT-PCR and western blot analyses further supported the clinical sample findings. CONCLUSION: The present data support the notion that the overexpression of ADAMTS4 in CRC might be useful as a non-invasive biomarker for detecting CRC in patients.


Asunto(s)
Proteína ADAMTS4/análisis , Neoplasias Colorrectales/patología , Anciano , Análisis de Varianza , Biomarcadores de Tumor , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/análisis , Valores de Referencia , Regulación hacia Arriba
10.
Braz J Med Biol Res ; 53(3): e9201, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32130294

RESUMEN

Methylophiopogonanone A (MO-A), a homoisoflavonoid extracted from Ophiopogon japonicus, has been shown to attenuate myocardial apoptosis and improve cerebral ischemia/reperfusion injury. However, the hypolipidemic effects remain unknown. This study was performed to investigate a potential hypolipidemic effect of MO-A in hyperlipidemia rats, as well as its underlying mechanism of action. A rat model of hyperlipidemia was induced by a high-fat diet (HFD). Animals were randomly divided into three groups (n=8/group): normal control group (NC), HFD group, and HFD+MO-A (10 mg·kg-1·d-1) treatment group. The effects of MO-A on serum lipids, body weight, activity of lipoprotein metabolism enzyme, and gene expression of lipid metabolism were evaluated in HFD-induced rats. In HFD-induced rats, pretreatment with MO-A decreased the body weight gain and reduced serum and hepatic lipid levels. In addition, pretreatment with MO-A improved the activities of lipoprotein lipase and hepatic lipase in serum and liver, down-regulated mRNA expression of acetyl CoA carboxylase and sterol regulatory element-binding protein 1c, and up-regulated mRNA expression of low-density lipoprotein receptor and peroxisome proliferator-activated receptor α in the liver. Our results indicated that MO-A showed strong ability to ameliorate the hyperlipidemia in HFD-induced rats. MO-A might be a potential candidate for prevention of overweight and dyslipidemia induced by HFD.


Asunto(s)
Benzodioxoles/farmacología , Dieta Alta en Grasa , Hiperlipidemias/prevención & control , Isoflavonas/farmacología , Metabolismo de los Lípidos , Ophiopogon/química , Animales , Benzodioxoles/aislamiento & purificación , Western Blotting , Modelos Animales de Enfermedad , Heces/química , Hiperlipidemias/metabolismo , Isoflavonas/aislamiento & purificación , Lípidos/análisis , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa
11.
Mem Inst Oswaldo Cruz ; 115: e190357, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32130369

RESUMEN

BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.


Asunto(s)
Virus del Dengue/enzimología , Glucógeno Sintasa Quinasas/antagonistas & inhibidores , Glucógeno Sintasa Quinasas/fisiología , Replicación Viral/fisiología , Aedes/citología , Animales , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral , Microscopía Fluorescente , Fosforilación/fisiología , Transducción de Señal
12.
Braz Oral Res ; 34: e006, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32022225

RESUMEN

Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Asunto(s)
Ameloblastos/citología , Células Madre Pluripotentes Inducidas/citología , Transducción de Señal/fisiología , Proteína Smad1/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Western Blotting , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero , Técnica del Anticuerpo Fluorescente , Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad1/análisis , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología
13.
Arq Neuropsiquiatr ; 78(1): 21-27, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32074185

RESUMEN

OBJECTIVE: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. METHODS: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. RESULTS: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. CONCLUSIONS: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.


Asunto(s)
Ácido Abscísico/farmacología , Analgésicos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Médula Espinal/metabolismo , Animales , Western Blotting , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Quinasas MAP Reguladas por Señal Extracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/farmacología , Masculino , Ratas Wistar , Valores de Referencia , Reproducibilidad de los Resultados , Médula Espinal/efectos de los fármacos , Factores de Tiempo
14.
Allergol. immunopatol ; 48(1): 8-17, ene.-feb. 2020. ilus
Artículo en Inglés | IBECS | ID: ibc-186586

RESUMEN

Introduction and objectives: LRBA deficiency is caused by loss of LRBA protein expression, due to either homozygous or compounds heterozygous mutations in LRBA. LRBA deficiency has been shown to affect vesicular trafficking and autophagy. To date, LRBA has been observed in the cytosol, Golgi apparatus and some lysosomes in LPS-stimulated murine macrophages. The objectives of the present study were to study the LRBA localization in organelles involved in vesicular traffic, phagocytosis, and autophagy in mononuclear phagocytes (MP). Materials and methods: We analyzed LRBA colocalization with different endosomes markets using confocal microscopy in MP. We used the autophagy inhibitors to determine the role of LRBA in formation, maturation or degradation of the autophagosome. Results: LRBA intracellular trafficking depends on the activity of the GTPase ADP ribosylation factor-1 (ARF) in MP. LRBA was identified in early, late endosomes but did not colocalize strongly with lysosomal markers. Although LRBA appears not to be recruited during the phagocytic cargo uptake, it greatly colocalized with the microtubule-associated protein 1A/1B-light chain 3 (LC3) under a steady state and this decreased after the induction of autophagy flux. Although the use of inhibitors of lysosome fusion did not restore the LRBA/LC3 colocalization, inhibitors of either early to late endosomes trafficking or PI3K pathway did. Conclusions: Taken together, our results show that LRBA is located in endomembrane system vesicles, mainly in the early and late endosomes. Although LRBA appears not to be involved in the phagocytic uptake, it is recruited in the early steps of the autophagy flux


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Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Membrana Celular/metabolismo , Lipopolisacáridos/farmacología , Síndromes de Inmunodeficiencia/complicaciones , Diferenciación Celular , Endosomas , Microscopía Confocal/métodos , Autofagosomas , Factores de Ribosilacion-ADP , Fagocitosis , Western Blotting , Sistema Mononuclear Fagocítico
15.
Acta Cir Bras ; 34(12): e201901202, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32049183

RESUMEN

PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Caryophyllaceae/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Extractos Vegetales/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Inmunohistoquímica , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/análisis , Fosfatidilinositol 3-Quinasas/análisis , Extractos Vegetales/química , Ratas Sprague-Dawley , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/análisis , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/efectos de los fármacos , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Tiempo
16.
Artículo en Inglés | MEDLINE | ID: mdl-32062365

RESUMEN

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.


Asunto(s)
Fragmentos Fab de Inmunoglobulinas/análisis , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fc de Inmunoglobulinas/análisis , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulinas/metabolismo , Sulfato de Amonio/química , Animales , Western Blotting , Pollos , Cromatografía por Intercambio Iónico , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulinas/química , Papaína/metabolismo
17.
Life Sci ; 245: 117364, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-32001263

RESUMEN

AIMS: To investigate the impact of microRNA target SNPs (mirSNPs) and their interaction with miRNAs on important drug-metabolizing enzymes, transporters and target genes for prediction of clopidogrel drug response in cardiovascular disease individuals. MAIN METHODS: A prospective cross-sectional study was conducted on 292 individuals undergoing clopidogrel drug therapy. All the enrolled participants were administered 300 mg loading dose followed by 75 mg dose of maintenance therapy. Platelet aggregations were measured before administration of the loading dose and 2 h post fifth day dose of clopidogrel maintenance therapy. Clopidogrel carboxylic acid metabolite from plasma and urine were analyzed post maintenance therapy using the RP-HPLC method. Genotyping of mirSNP's shortlisted through in silico analysis was performed by tetra ARMS PCR and validated by Sanger DNA sequencing. The levels of selected miRNAs were estimated by the TaqMan-PCR assay. Functional validation of mirSNPs was performed in HepG2 cells after transfecting with the selected gene and miRNA mimics. Protein expressions were analyzed by western blot. KEY FINDINGS: 23% of enrolled individuals showed resistance to clopidogrel therapy. Out of 13 mirSNP's analyzed, CYP2C19 rs4244285 was associated with clopidogrel drug resistance and clopidogrel carboxylic acid metabolite in urine and plasma. hsa-miR-1343-3p and hsa-miR-6783-3p levels were significantly high in individuals with CYP2C19 rs4244285 mutant genotype and these miRNAs down-regulated the protein expression of CYP2C19. SIGNIFICANCE: We demonstrated the role of coding mirSNP (rs4244285) in the regulation of the CYP2C19 gene through miRNAs and its implications to clopidogrel drug response prediction in the Indian population.


Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Clopidogrel/farmacología , Citocromo P-450 CYP2C19/genética , MicroARNs/genética , Agregación Plaquetaria/efectos de los fármacos , Polimorfismo de Nucleótido Simple/genética , Western Blotting , Clopidogrel/metabolismo , Clopidogrel/uso terapéutico , Estudios Transversales , Citocromo P-450 CYP2C19/metabolismo , Resistencia a Medicamentos/genética , Femenino , Células Hep G2 , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Secuencia de ADN
18.
Life Sci ; 245: 117365, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-32001267

RESUMEN

AIMS: Hyperglycemia in combination with oxidative stress plays a significant pathophysiological role in diabetic testicular dysfunction, often leading to infertility. Activation of Toll-like receptor 4 (TLR4) has been reported to mediate oxidative stress during diabetes. However, engagement of the TLR4 signaling pathway in diabetic testicular dysfunction has not been previously explored. Herein, we investigated the role of TLR4 in reactive oxygen species (ROS) production and in the phosphorylation status of ERK1/2 in primary Leydig cells exposed to high glucose and in testis isolated from diabetic rats. MAIN METHODS: Testicular levels of TLR4 and phospho-ERK1/2 were determined by Western blotting. ROS production was detected with a fluorescent probe. Additionally, primary Leydig cells were exposed to normal (5.5 mmol/l) or elevated (33 mmol/l) glucose concentrations and treated with or without a TLR4 inhibitor, CLI095 (10-5 mol/l) for 24 h, followed by evaluation of TLR4 and phospho-ERK1/2 expression levels by Western blotting and immunofluorescence staining, respectively. KEY FINDINGS: We show that high glucose induces the expression of TLR4 in Leydig cells. Additionally, we demonstrate that blockade of this receptor in this cell population reduces oxidative stress and restores the levels of phospho-ERK1/2. SIGNIFICANCE: Our findings provide new insight into TLR4 interaction with ROS and MEK/ERK pathway in Leydig cells exposed to high glucose and present a rationale for the development of new therapeutics for diabetic testicular dysfunction.


Asunto(s)
Hiperglucemia/metabolismo , Células Intersticiales del Testículo/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estrés Oxidativo , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo
19.
Life Sci ; 245: 117368, 2020 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-32001270

RESUMEN

AIMS: Amino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs. MAIN METHODS: An in vivo model of I/R injury was tested in control, mTOR+/+, and mTOR+/-. Mice were randomly assigned to receive BCAAs, rapamycin, or BCAAs + rapamycin. Furthermore, isolated cardiomyocytes were subjected to simulated ischemia and cell death was quantified. Biochemical and mitochondrial swelling assays were also performed. KEY FINDINGS: Mice treated with BCAAs had a significant reduction in infarct size as a percentage of the area at risk compared to controls (34.1 ± 3.9% vs. 44.7 ± 2.6%, P = 0.001), whereas mice treated with the mTOR inhibitor rapamycin were not protected by BCAA administration (42.2 ± 6.5%, vs. control, P = 0.015). This protection was not detected in our hetero knockout mice of mTOR. Western blot analysis revealed no change in AKT signaling whereas activation of mTOR was identified. Furthermore, BCAAs prevented swelling which was reversed by the addition of rapamycin. In myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA-induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). SIGNIFICANCE: BCAA treatment exhibits a protective effect in myocardial I/R injury and that mTOR plays an important role in this preconditioning effect.


Asunto(s)
Aminoácidos de Cadena Ramificada/uso terapéutico , Cardiotónicos/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Animales , Western Blotting , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Wistar , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo
20.
Life Sci ; 246: 117382, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32004509

RESUMEN

Our preliminary research revealed that metformin, a classic anti-diabetic drug, could rescue Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells in vitro, but the molecular mechanism remains to be explored. In the study, Human Renal Cortical Epithelial Cells (HRCEpiC) and Human Renal Proximal Tubular Epithelial Cells (HRPTEpic) were challenged with high glucose with or without metformin pre-treatment to monitor Parkin mRNA and protein expression level change. PRKN gene knockdown was performed by lentiviral-based shRNA delivery. Cell viability, apoptosis and mitophagy were monitored after treatment. Mitochondrial damage was evaluated by analyzing mitochondrial permeability transition pore opening, membrane potential change, mitochondrial superoxide accumulation and cytochrome C release. Protein levels of activating transcription factor 4 (ATF4), p53 phospho-Ser15, IκBα phosphor-Ser32, IKKα phosphor-Ser176/180 in whole cell lysate and nuclear entry of p50/p65 were assessed by western blot. Okadaic acid was used to inhibit protein phosphatase 2A (PP2A). The data suggested high glucose challenge significantly reduced PRKN gene expression, mitophagy, mitochondria integrity and cell viability in vitro, which was rescued by metformin co-treatment. The effects of metformin were crippled by PRKN gene knockdown. Metformin increased PRKN gene transcription while reducednuclear factor kappa B (NF-κB) activation but not that of p53 or ATF4. Inhibiting PP2A weakened NF-κB inhibition and PRKN induction by metformin in high glucose-challenged cells, reducing its mitochondrial protective and cytoprotective effect. So, we concluded thatmetformin protects human renal epithelial cells from high glucose-induced apoptosis by restoring Parkin protein expression and mitophagy via PP2A activation and NF-κB inhibition.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Hipoglucemiantes/farmacología , Riñón/efectos de los fármacos , Metformina/farmacología , Mitofagia/efectos de los fármacos , FN-kappa B/metabolismo , Proteína Fosfatasa 2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Western Blotting , Nefropatías Diabéticas/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Células Epiteliales/metabolismo , Glucosa/farmacología , Humanos , Riñón/citología , Riñón/metabolismo , Mitocondrias/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa
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