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1.
Drug Des Devel Ther ; 13: 3291-3306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571831

RESUMO

Objectives: This study was performed to evaluate the effects of muscone on the proliferation, migration and differentiation of human gingival mesenchymal stem cells (GMSCs) and to explore the relevant mechanisms. Materials and methods: We performed studies to determine the effects and mechanisms of muscone on GMSC proliferation, migration and differentiation. We conducted CCK-8, colony formation, transwell chamber, scratch wound, alkaline phosphatase (ALP) staining and activity, and alizarin red and oil red O staining assays, as well as real-time quantitative polymerase chain reaction (qRT-PCR), to ascertain the effects of muscone on GMSC proliferation, migration and differentiation in vitro. The mechanism by which muscone influences the osteogenic and adipogenic differentiation of GMSCs was elucidated by qRT-PCR and Western blotting. Results: We found that muscone significantly promoted GMSC proliferation, chemotaxis, wound healing and fat droplet formation and inhibited ALP activity and mineral deposition. Notably, we observed that the Wnt/ß-catenin pathway was closely related to the ability of muscone to inhibit the osteogenic differentiation and promote the adipogenic differentiation of GMSCs. The effect of muscone on the multidirectional differentiation capacity of GMSCs was significantly reversed by the agonist lithium chloride through the Wnt/ß-catenin signaling pathway. Conclusion: Muscone effectively increased the proliferation and migration, promoted the adipogenic differentiation and inhibited the osteogenic differentiation of GMSCs by inhibiting the Wnt/ß-catenin signaling pathway. These results may provide a theoretical basis for the application of GMSCs and muscone in tissue engineering and regenerative medicine.


Assuntos
Adipogenia/efeitos dos fármacos , Cicloparafinas/farmacologia , Gengiva/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Adipogenia/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Humanos , Cloreto de Lítio/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
2.
Int J Nanomedicine ; 14: 7475-7488, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571859

RESUMO

Background: Wear particle-induced inflammatory osteolysis and the consequent aseptic loosening constitute the leading reasons for prosthesis failure and revision surgery. Several studies have demonstrated that the macrophage polarization state and immune response play critical roles in periprosthetic osteolysis and tissue repair, but the immunomodulatory role of lithium chloride (LiCl), which has a protective effect on wear particle-induced osteolysis by suppressing osteoclasts and attenuating inflammatory responses, has never been investigated. Methods: In this work, the immunomodulatory capability of LiCl on titanium (Ti) nanoparticle-stimulated transformation of macrophage phenotypes and the subsequent effect on osteogenic differentiation were investigated. We first speculated that LiCl attenuated Ti nanoparticle-stimulated inflammation responses by driving macrophage polarization and generating an immune micro-environment to improve osteogenesis. Furthermore, a metal nanoparticle-stimulated murine air pouch inflammatory model was applied to confirm this protective effect in vivo. Results: The results revealed that metal nanoparticles significantly activate M1 phenotype (proinflammatory macrophage) expression and increase proinflammatory cytokines secretions in vitro and in vivo, whereas LiCl drives macrophages to the M2 phenotype (anti-inflammatory macrophage) and increases the release of anti-inflammatory and bone-related cytokines. This improved the osteogenic differentiation capability of rat bone marrow mesenchymal stem cells (rBMSCs). In addition, we also provided evidence that LiCl inhibits the phosphorylation of the p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways in wear particle-treated macrophages. Conclusion: LiCl has the immunomodulatory effects to alleviate Ti nanoparticle-mediated inflammatory reactions and enhance the osteogenic differentiation of rBMSCs by driving macrophage polarization. Thus, LiCl may be an effective therapeutic alternative for preventing and treating wear debris-induced inflammatory osteolysis.


Assuntos
Fatores Imunológicos/farmacologia , Inflamação/patologia , Cloreto de Lítio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/química , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Ratos
3.
Fish Physiol Biochem ; 45(6): 1953-1961, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31401708

RESUMO

Glycogen synthase kinase-3ß (GSK3ß) is a serine/threonine kinase involved in the regulation of embryonic development, glycogen metabolism, protein synthesis, mitosis, and apoptosis. To understand the role of GSK3ß in hepatic lipid accumulation of Schizothorax prenanti, we used lithium chloride (LiCl), a GSK3ß inhibitor, to inhibit the expression and activity of GSK3ß. LiCl increased levels of phosphorylation of GSK3ß (Ser9) and decreased the protein level of GSK3ß. Plasma TG, TC, and LDL-C levels were greatly decreased after LiCl treatment. Additionally, GSK3ß inhibition significantly reduced the levels of hepatic triglyceride (TG) and decreased the expression of lipogenesis-related genes in liver. Interestingly, LiCl decreased levels of phosphorylation of STAT3 (Tyr705), and then inhibited the activity of STAT3. These results indicate that in vivo LiCl treatment, which inhibited GSK3ß activity, effectively decreased hepatic lipid accumulation through STAT3 in Schizothorax prenanti.


Assuntos
Cyprinidae/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Metabolismo dos Lipídeos , Cloreto de Lítio/farmacologia , Fígado/metabolismo , Animais , Lipídeos/sangue , Lipogênese , Fosforilação , Distribuição Aleatória , Fator de Transcrição STAT3/metabolismo
4.
Mol Cell Biochem ; 461(1-2): 127-139, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31352609

RESUMO

Constitutive androstane receptor (CAR) is a xenobiotic nuclear receptor known to regulate genes involved in key physiological processes like drug metabolism, maintenance of energy homeostasis, and cell proliferation. Owing to the diverse regulatory roles played by the receptor, it is critical to understand the precise cellular signals that dictate functional dynamics of CAR. With the objective of exploring the hitherto unknown regulatory pathways modulating CAR, we subjected the CAR protein sequence to a kinase prediction tool and identified several kinases recognizing CAR as a substrate. Using fluorescence live cell imaging and specific inhibitors it was observed that CAR functions under the regulation of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) signaling cascade. Additionally, insulin-like growth factor 1 (IGF1)-mediated inhibition of GSK3 also induced nuclear translocation of CAR linking CAR to the Akt signaling pathway. Identification of T38 residue of CAR as the GSK3 target site further substantiated our observations. Taking cues from these findings, we propose a hypothetical model elucidating the GSK3-mediated regulation of CAR dynamics through the involvement of Akt pathway. Further research into this area is expected to provide novel therapeutic targets in disease conditions like type 2 diabetes and hepatocellular carcinoma.


Assuntos
Núcleo Celular/metabolismo , Proteínas Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Ligantes , Cloreto de Lítio/farmacologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/química
5.
Toxicol Lett ; 313: 11-18, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220555

RESUMO

Previous study reported that either selective GSK-3ß inhibitor or up-regulating autophagy can alleviate cisplatin-induced ototoxicity. Other studies indicate that the activity of GSK-3ß is closely associated with the autophagy level. The purpose of this study is to primarily explore the role of autophagy in the alleviation effect of GSK-3ß inhibition on cisplatin-induced ototoxicity in vivo and in vitro. We observed the autophagy changes induced by GSK-3ß inhibitor in outer hair cells (OHCs) in a cisplatin-induced ototoxicity rat model. In addition, autophagy inhibitor 3-MA was used in vitro experiments to observe the influence of autophagy inhibition on the cell protection effect due to GSK-3ß inactivation. The relationship among autophagy, GSK-3ß and cell damage were inferred. Negative regulation of GSK-3ß significantly enhanced autophagy and alleviated cisplatin-induced hearing loss, OHC death in vivo and apoptosis in vitro. The autophagy inhibitor 3-MA inverted the protective effect of negative regulation of GSK-3ß. These results indicated that enhancing autophagy may be a key downstream effect of GSK-3ß inhibition in the alleviation of cisplatin-induced ototoxicity both in vivo and in vitro.


Assuntos
Autofagia/efeitos dos fármacos , Cisplatino , Otopatias/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Ciliadas Auditivas/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Animais , Fadiga Auditiva/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Otopatias/induzido quimicamente , Otopatias/enzimologia , Otopatias/patologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Células Ciliadas Auditivas/enzimologia , Células Ciliadas Auditivas/patologia , Masculino , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
6.
Food Chem ; 296: 56-62, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31202306

RESUMO

Autophagy, a mechanism of recycling intracellular constituents, favors plant growth, especially under nutrient starvation. However, autophagy's role in regulating postharvest fruit senescence is unclear. Here, effects of the autophagy inhibitor hydroxychloroquine (HCQ) and activator LiCl on postharvest jujube fruit senescence were investigated. HCQ significantly reduced weight loss and decay incidence, and enhanced firmness compared with those of the control. LiCl had the opposite effects. Protein oxidation and H2O2 increased significantly in LiCl-treated compared with HCQ-treated fruit. The contents of vitamin C, total thiol, and phenolics, and total antioxidant capacity and DPPH-radical scavenging capacity, followed the order: HCQ > control > LiCl. The HCQ-mediated reduction in fruit respiration was significantly enhanced by ATP and partly reversed by 2,4-dinitrophenol, a mitochondrial uncoupler. Thus, jujube fruit senescence may be regulated by autophagy and the antioxidant capacity. A mechanism of autophagy-mediated postharvest fruit senescence involving mitochondrial biogenesis and respiration was proposed.


Assuntos
Autofagia/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Nutrientes/metabolismo , Ziziphus/metabolismo , Antioxidantes/química , Ácido Ascórbico/química , Frutas/efeitos dos fármacos , Frutas/metabolismo , Peróxido de Hidrogênio/análise , Hidroxicloroquina/química , Cloreto de Lítio/farmacologia , Nutrientes/química , Fenóis/química , Temperatura , Ziziphus/efeitos dos fármacos
7.
Med Sci Monit ; 25: 4041-4050, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31147532

RESUMO

BACKGROUND We synthetized a 3D printed poly-ε-caprolactone (PCL) scaffold with polydopamine (PDA) coating and lithium chloride (LiCl) deposition for cartilage tissue engineering and analyzed its effect on promoting rabbit bone marrow mesenchymal stem cells (rBMSC) chondrogenesis in vitro. MATERIAL AND METHODS PCL scaffolds were prepared by 3D printing with a well-designed CAD digital model, then modified by PDA coating to produce PCL-PDA scaffolds. Finally, LiCl was deposited on the PDA coating to produce PCL-PDA-Li scaffolds. The physicochemical properties, bioactivity, and biocompatibility of PCL-PDA-Li scaffolds were accessed by comparing them with PCL scaffolds and PCL-PDA scaffolds. RESULTS 3D PCL scaffolds exhibited excellent mechanical integrity as designed. PDA coating and LiCl deposition improved surface hydrophilicity without sacrificing mechanical strength. Li⁺ release was durable and ion concentration did not reach the cytotoxicity level. This in vitro study showed that, compared to PCL scaffolds, PCL-PDA and PCL-PDA-Li scaffolds significantly increased glycosaminoglycan (GAG) formation and chondrogenic marker gene expression, while PCL-PDA-Li scaffolds showed far higher rBMSC viability and chondrogenesis. CONCLUSIONS 3D printed PCL-PDA-Li scaffolds promoted chondrogenesis in vitro and may provide a good method for lithium administration and be a potential candidate for cartilage tissue engineering.


Assuntos
Cloreto de Lítio/farmacologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Medula Óssea , Caproatos/farmacologia , Cartilagem/metabolismo , Condrogênese/fisiologia , Indóis/farmacologia , Lactonas/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Poliésteres/química , Polímeros/farmacologia , Impressão Tridimensional , Coelhos , Regeneração/fisiologia
8.
PLoS One ; 14(6): e0217458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31216290

RESUMO

Rats not only avoid ingesting a substance associated with LiCl toxicosis, but they display rejection reflexes (e.g., gapes) to its taste; this latter response is thought to reflect disgust or taste aversion. Prior work has shown that rats also avoid consuming foods/fluids associated with other adverse gastrointestinal (GI) effects like lactose indigestion but without the concomitant change in oromotor responses (taste reactivity; TR) indicative of aversion. Because of interpretive limitations of the methods used in those studies, we revisited the taste aversion-avoidance distinction with a design that minimized non-treatment differences among groups. Effects on intake and preference (Experiments 1a, 1b, and 2), as well as consummatory (TR, Experiment 1a and 1b) and appetitive (Progressive Ratio, Experiment 2) behaviors to the taste stimulus were assessed after training. In both experiments, rats were trained to associate 0.2% saccharin (CS) with intraduodenal infusions of LiCl, Lactose, or NaCl control. Rats trained with 18% lactose, 0.3 and 1.5 mEq/kg dose of LiCl subsequently avoided the taste CS in post-training single-bottle intake tests and two-bottle choice tests. However, only those trained with 1.5 mEq/kg LiCl displayed post-conditioning increases in taste CS-elicited aversive TR (Experiment 1a and 1b). This dose of LiCl also led to reductions in breakpoint for saccharin. The fact that conditioned avoidance is not always accompanied by changes in other common appetitive and/or consummatory indices of ingestive motivation further supports a functional dissociation between these processes, and highlights the intricacies of visceral influences on taste-guided ingestive motivation.


Assuntos
Aprendizagem da Esquiva/fisiologia , Modelos Biológicos , Percepção Gustatória/fisiologia , Paladar/fisiologia , Animais , Agentes Aversivos/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Sacarina/farmacologia , Percepção Gustatória/efeitos dos fármacos
9.
Neurosci Lett ; 708: 134349, 2019 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-31238129

RESUMO

The neuro-protective effects of rubidium and lithium as alkali metals have been reported for different central nervous system dysfunctions including mania and depression. The aim of this study was evaluating as well as comparing the effects of rubidium chloride (RbCl) and lithium chloride (LiCl) on different seizures paradigms in mice and determining the involvement of NMDA receptors and nitrergic pathway. To assess the seizures threshold, animals received intravenous pentylenetetrazole (PTZ, 0.5%; 1 mL/min). Male NMRI mice (6-8 weeks) received intraperitoneal (i.p.) injections of different doses of RbCl and LiCl. Doses greater than 10 mg/kg of RbCl showed a significant anticonvulsant activity 60 min after administration; the anticonvulsant effects of LiCl was observed at the doses more than 5 mg/kg and after 30 min in PTZ-induced seizure threshold. But, RbCl (10, 20 mg/kg, i.p) or LiCl (5, 10 mg/kg, i.p) injection did not induce protection against maximal electroshock (MES) or intraperitoneal injection of PTZ lethal dose (80 mg/kg)-induced seizure models. Pre-treatment with L-NAME (non-selective nitric oxide synthase (NOS) inhibitor, 10 mg/kg; i.p.) and 7-nitroindazole (selective neuronal NOS inhibitor, 30 mg/kg; i.p.) enhanced the anticonvulsive effects of both RbCl (5 mg/kg, i.p.) and LiCl (1 mg/kg, i.p.) in PTZ-induced seizure threshold model. Injection of MK-801 (NMDA receptor antagonist, 0.05 mg/kg; i.p.) before RbCl (5 mg/kg, i.p.; P < 0.001) and LiCl (1 mg/kg, i.p.; P < 0.001) administration increased the anti-seizure activity. But, treatment with L-arginine (precursor of nitric oxide, 100 mg/kg; i.p.) decreased the seizure threshold of both RbCl (20 mg/kg, i.p.; P < 0.001) and LiCl (10 mg/kg, i.p.; P < 0.001). Measurement of nitrite levels in hippocampus of animals revealed a remarkable reduction after treatment with RbCl (20 mg/kg, i.p; P < 0.05) and LiCl (10 mg/kg, i.p; P < 0.01). To conclude, rubidium may protect central nervous system against seizures in PTZ-induced seizures threshold model through NMDA/nitrergic pathways with a similarity to lithium effects in mice.


Assuntos
Anticonvulsivantes/farmacologia , Cloretos/farmacologia , Ácido Glutâmico/metabolismo , Cloreto de Lítio/farmacologia , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Rubídio/farmacologia , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/uso terapêutico , Cloretos/uso terapêutico , Cloreto de Lítio/uso terapêutico , Masculino , Camundongos , Fármacos Neuroprotetores/uso terapêutico , Pentilenotetrazol , Receptores de N-Metil-D-Aspartato/metabolismo , Rubídio/uso terapêutico , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Transdução de Sinais
10.
Int J Mol Sci ; 20(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242642

RESUMO

The ketogenic diet (KD), a high-fat/low-carbohydrate/adequate-protein diet, has been proposed as a treatment for a variety of diseases, including cancer. KD leads to generation of ketone bodies (KBs), predominantly acetoacetate (AcAc) and 3-hydroxy-butyrate, as a result of fatty acid oxidation. Several studies investigated the antiproliferative effects of lithium acetoacetate (LiAcAc) and sodium 3-hydroxybutyrate on cancer cells in vitro. However, a critical point missed in some studies using LiAcAc is that Li ions have pleiotropic effects on cell growth and cell signaling. Thus, we tested whether Li ions per se contribute to the antiproliferative effects of LiAcAc in vitro. Cell proliferation was analyzed on neuroblastoma, renal cell carcinoma, and human embryonic kidney cell lines. Cells were treated for 5 days with 2.5, 5, and 10 mM LiAcAc and with equimolar concentrations of lithium chloride (LiCl) or sodium chloride (NaCl). LiAcAc affected the growth of all cell lines, either negatively or positively. However, the effects of LiAcAc were always similar to those of LiCl. In contrast, NaCl showed no effects, indicating that the Li ion impacts cell proliferation. As Li ions have significant effects on cell growth, it is important for future studies to include sources of Li ions as a control.


Assuntos
Acetoacetatos/farmacologia , Lítio/farmacologia , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cloretos/farmacologia , Expressão Gênica , Humanos , Cloreto de Lítio/farmacologia
11.
PLoS One ; 14(5): e0209812, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31083677

RESUMO

Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium.


Assuntos
Materiais Biocompatíveis/metabolismo , Carpóforos/fisiologia , Fungos/fisiologia , Agaricales , Biomarcadores , Inibidores Enzimáticos/farmacologia , Carpóforos/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Cloreto de Lítio/farmacologia , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento
12.
Nucleic Acids Res ; 47(13): 6783-6795, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31066439

RESUMO

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.


Assuntos
DNA de Cadeia Simples/química , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , Pegada de DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Recombinante/química , Cloreto de Lítio/farmacologia , Microscopia de Força Atômica , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico/efeitos dos fármacos , Hibridização de Ácido Nucleico , Plasmídeos/genética , RNA Longo não Codificante/química , Proteínas de Schizosaccharomyces pombe/genética , Transcrição Genética
13.
Curr Med Sci ; 39(1): 28-36, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30868488

RESUMO

In this study, the hypothesis that Wnt/ß-catenin pathway is involved in the arterial calcification by regulating the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) system was tested. The ß-catenin expression was measured in the warfarin-induced calcified arteries and the osteoblast-like cells differentiating from smooth muscle cells (SMCs) by immunohistochemistry and Western blotting. The Wnt/ß-catenin pathway was activated or inhibited by lithium chloride (LiCl) or dickkopf 1 (DKK1) in vitro and in vivo. Then the calcification level was determined by von Kossa staining, Ca2+ content assay, and alkaline phosphatase (ALP) activity assay. The expression levels of osteocalcin, OPG and RANKL were detected by Western blotting or real-time PCR. The results showed that in calcified arteries and OBL cells, the activation of Wnt/ß-catenin pathway significantly enhanced the calcification as evidenced by increased von Kossa stains, Ca2+ contents, ALP activities, and osteocalcin expression levels (P<0.05), and it promoted the RANKL expression (P<0.05), but slightly affected the OPG expression. These results indicated that the activation of Wnt/ß-catenin pathway worsens the arterial calcification, probably by promoting the RANKL expression.


Assuntos
Músculo Liso Vascular/irrigação sanguínea , Osteoprotegerina/genética , Ligante RANK/genética , Calcificação Vascular/metabolismo , Varfarina/efeitos adversos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Cloreto de Lítio/farmacologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Ratos , Calcificação Vascular/induzido quimicamente , Calcificação Vascular/genética , beta Catenina/metabolismo
14.
Curr Med Sci ; 39(1): 75-80, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30868494

RESUMO

Nowadays, the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head (ONFH). Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level. Glycogen synthase kinase 3ß (GSK3ß) is an important regulator of cellular differentiation and apoptosis pathway, which can modulate the balance between osteoblasts and osteoclasts. Several studies have reported about its function in osteoporosis, but little is known about it in osteonecrosis. In our study, lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model. The phosphorylation of GSK3ß Ser-9 was decreased in the model. Western blotting examination of ß-catenin, Bcl-2, Bax and caspase-3 revealed that the osteoblasts were apoptotic. In dexamethasone (Dex)-incubated primary osteoblasts, the expression profile of GSK3ß phosphorylation and apoptotic factors were consistent with those in the rat ONFH model. To further investigate the regulation of osteonecrosis caused by GSK3ß, the expression and function of GSK3ß were inhibited in Dex-incubated primary osteoblasts. The knockdown of GSK3ß by siRNA decreased the expression of Bax and cleaved caspase-3, but increased Bcl-2 and ß-catenin. On the other hand, selective inhibition of GSK3ß function by LiCl counteracted the activation of caspase-3 induced by Dex. Our work is the first study about the GSK3ß phosphorylation in ONFH, and provides evidence for further therapeutic methods.


Assuntos
Necrose da Cabeça do Fêmur/induzido quimicamente , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Osteoartrite/induzido quimicamente , Osteoblastos/citologia , Serina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Dexametasona , Modelos Animais de Doenças , Necrose da Cabeça do Fêmur/genética , Necrose da Cabeça do Fêmur/metabolismo , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/efeitos adversos , Cloreto de Lítio/farmacologia , Metilprednisolona/efeitos adversos , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoblastos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Regulação para Cima/efeitos dos fármacos
15.
Breast Cancer Res ; 21(1): 37, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845991

RESUMO

BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Conjuntos de Dados como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Cloreto de Lítio/farmacologia , Cloreto de Lítio/uso terapêutico , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Via de Sinalização Wnt
16.
Int J Mol Med ; 43(5): 2075-2085, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864678

RESUMO

von Willebrand factor C and EGF domain­containing protein (URG11), a cell growth regulator, is involved in the progression of a variety of types of cancer, including prostate cancer (Pca). However, the functions of the URG11 gene in Pca cells require in­depth investigation. The mRNA and protein levels of URG11 were measured by reverse transcription quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Cell Counting kit­8 (CCK­8), wound­healing and Transwell assays were used to detect cell viability, migration and invasion, respectively. Apoptosis and cell cycle analyses were performed using flow cytometry. The mRNA and protein expression levels of epithelial (E)­cadherin, vimentin, α­smooth muscle actin (α­SMA), cyclin D1 and MYC proto­oncogene protein (c­Myc) were analyzed by RT­qPCR and western blot analysis. In the present study, the mRNA and protein levels of URG11 were markedly upregulated in Pca cell lines compared with those in the normal prostate epithelial cell line. With functional experiments, the cell viability, migration and invasion of Pca cells were markedly promoted by URG11 overexpression. The cell cycle was effectively induced by URG11 and apoptosis was inhibited by the overexpression of URG11. Concomitantly, the epithelial marker E­cadherin was downregulated, and the mesenchymal markers vimentin and α­SMA were upregulated following URG11 overexpression. By contrast, genetic knockout of URG11 elicited the opposite effects. The present study also identified that the downstream effector genes of the Wnt/ß­catenin signal pathway, cyclin D1 and c­Myc, were increased following the overexpression of endogenous URG11, which are known to regulate cell proliferation. In addition, the Wnt/ß­catenin inhibitor FH535 ameliorated the promotive effects of URG11 on LNCaP cells viability, migration and invasion, and the Wnt/ß­catenin agonist LiCl reversed the inhibitory effects of siURG11 in LNCaP cells on cell viability, migration and invasion. The present study demonstrated that URG11 served an oncogenic role in the development of Pca cells and provided evidence that URG11 has potential as a novel therapeutic target in Pca.


Assuntos
Apoptose , Transativadores/metabolismo , Actinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Caderinas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ciclina D1/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cloreto de Lítio/farmacologia , Masculino , Invasividade Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Sulfonamidas/farmacologia , Transativadores/genética , Vimentina/metabolismo
17.
Neurol Res ; 41(6): 577-583, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30879425

RESUMO

OBJECTIVE: Nowadays, there seems to be no decisive way for treatment of spinal cord injury (SCI).Extensive cell death (apoptosis and necrosis) occurring in SCI can cause considerable progressive sensorimotor disabilities. Preventing cell death by improving endogenous regenerative capability could an effective strategy for the treatment of SCI. This study was designed to evaluate the effects of lithium chloride (LiCl) on the cell survival through overexpression of BDNF and NT3 mRNA level and their receptors in the contusion rat models. METHODS: Rats were randomly divided into four experimental groups (eight rats/group) including: contused animals (the non-treatment group); contused animals (the control group) which received laminectomy; contused animals received normal saline (vehicle)and contused animals received intraperitoneal injection of 20 mg/kg LiCl three days after surgery. Injection continued for 14 days as treatment. Basso, Beattie, Bresnahan (BBB) rating scale was used to assess the motor function of the rats. To evaluate the histopathological and gene expression analysis, rats were sacrificed 28 days after surgery. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to obtain the relative levels of mRNA for BDNF, NT3 and their receptors. RESULTS: The results showed LiCl ameliorates BBB scores via up-regulation of BDNF and TrkB receptors. Also, histological analysis showed that the numerical density per area of TUNEL- positive cells and the percentage of cavity significantly decreased in the LiCl-treated group. CONCLUSION: Our findings suggest that LiCl protects neural cells and effectively enhances locomotor function, which was done through up-regulation of endogenous BDNF expression in rats with SCI. ABBREVIATIONS: SCI: spinal cord injury; LiCl: lithium chloride; BDNF: Brain-derived neurotrophic factor; NT3: Neurotrophin-3; BBB: Basso, Beattie, Bresnahan; TrkB: Tropomyosin receptor kinase B; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Contusões/tratamento farmacológico , Cloreto de Lítio/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Atividade Motora/fisiologia , Fatores de Crescimento Neural/efeitos dos fármacos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia
18.
PLoS One ; 14(2): e0211426, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707732

RESUMO

The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1-16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1-16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/química , Fosfoproteínas Fosfatases/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cloreto de Lítio/farmacologia , Mutagênese , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
J Med Chem ; 62(2): 727-741, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30605343

RESUMO

Wnt/ß-catenin signaling pathway is implicated in the etiology and progression of metabolic disorders. Although lines of genetic evidence suggest that blockage of this pathway yields favorable outcomes in treating such ailments, few inhibitors have been used to validate the promising genetic findings. Here, we synthesized and characterized a novel class of triazole-based Wnt/ß-catenin signaling inhibitors and assessed their effects on energy metabolism. One of the top inhibitors, compound 3a, promoted Axin stabilization, which led to the proteasome degradation of ß-catenin and subsequent inhibition of the Wnt/ß-catenin signaling in cells. Treatment of hepatocytes and high fat diet-fed mice with compound 3a resulted in significantly decreased hepatic lipid accumulation. Moreover, compound 3a improved glucose tolerance of high fat diet-fed mice without noticeable toxicity, while downregulating the genes involved in the glucose and fatty acid anabolisms. The new inhibitors are expected to be further developed for the treatment of metabolic disorders.


Assuntos
Dieta Hiperlipídica , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Obesidade/etiologia , Triazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Teste de Tolerância a Glucose , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cloreto de Lítio/farmacologia , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/uso terapêutico , beta Catenina/metabolismo
20.
Int J Neuropsychopharmacol ; 22(4): 303-316, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649326

RESUMO

BACKGROUND: Adolescent methamphetamine exposure causes a broad range of neurobiological deficits in adulthood. Glycogen synthase kinase-3ß is involved in various cognitive and behavioral processes associated with methamphetamine exposure. This study aims to investigate the protective effects of the glycogen synthase kinase-3ß inhibitor lithium chloride on adolescent methamphetamine exposure-induced long-term alterations in emotion, cognition, behavior, and molecule and hippocampal ultrastructure in adulthood. METHODS: A behavioral test battery was used to investigate the protective effects of lithium chloride on adolescent methamphetamine exposure-induced long-term emotional, cognitive, and behavioral impairments in mice. Western blotting and immunohistochemistry were used to detect glycogen synthase kinase-3ß activity levels in the medial prefrontal cortex and dorsal hippocampus. Electron microscopy was used to analyze changes in synaptic ultrastructure in the dorsal hippocampus. Locomotor sensitization with a methamphetamine (1 mg/kg) challenge was examined 80 days after adolescent methamphetamine exposure. RESULTS: Adolescent methamphetamine exposure induced long-term alterations in locomotor activity, novel spatial exploration, and social recognition memory; increases in glycogen synthase kinase-3ß activity in dorsal hippocampus; and decreases in excitatory synapse density and postsynaptic density thickness in CA1. These changes were ameliorated by lithium chloride pretreatment. Adolescent methamphetamine exposure-induced working memory deficits in Y-maze spontaneous alternation test and anxiety-like behavior in elevated-plus maze test spontaneously recovered after long-term methamphetamine abstinence. No significant locomotor sensitization was observed after long-term methamphetamine abstinence. CONCLUSIONS: Hyperactive glycogen synthase kinase-3ß contributes to adolescent chronic methamphetamine exposure-induced behavioral and hippocampal impairments in adulthood. Our results suggest glycogen synthase kinase-3ß may be a potential target for the treatment of deficits in adulthood associated with adolescent methamphetamine abuse.


Assuntos
Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Disfunção Cognitiva/prevenção & controle , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Metanfetamina/farmacologia , Fatores Etários , Animais , Disfunção Cognitiva/induzido quimicamente , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Memória de Curto Prazo/efeitos dos fármacos , Camundongos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo
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