RESUMO
Obesity is accompanied by an increase in the size and the number of adipocytes. As adipocytes are thought to differentiate from pre-adipocytes, we postulate that non-adipogenic fibroblasts contribute to adipocyte formation under certain conditions such as obesity. We report for the first time that NIH-3T3 fibroblasts, which are generally considered to be non-adipogenic, can be converted into mature adipocytes by treatment with a defined hormone mixture comprising EGF (epidermal growth factor), HGF (hepatocyte growth factor), Dex (dexamethasone) and insulin. Furthermore, NIH-3T3 cells transplanted into obese immunodeficient NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice formed adipocytes in vivo. Interestingly, the mixture elicited conversion of NIH-3T3 cells directly into adipocytes without a preceding pre-adipocyte stage. Functional analysis revealed that each component of the mixture was necessary for the induction of adipogenesis, including Dex which inhibited the cell proliferation stimulated by EGF. Upon profiling the signalling pathways employed by EGF and HGF, we found STAT5 (signal transducer and activator of transcription 5) signalling to be activated, predominantly at the levels of both transcription and post-translational modification. Inhibition of the STAT5 pathway, either by genetic knockdown or a specific pharmacological agent, blocked adipogenesis in NIH-3T3 cells. Taken together, these data not only establish a newly recognized grouping of factors that can induce trans-differentiation of non-adipogenic fibroblasts into adipocytes, but also give us a greater understanding of obesity.
Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Dexametasona/administração & dosagem , Fator de Crescimento Epidérmico/administração & dosagem , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Fator de Crescimento de Hepatócito/administração & dosagem , Insulina/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células NIH 3T3 , Obesidade/metabolismo , Obesidade/patologia , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The present study was conducted to investigate whether Ginkgo biloba extract (EGb) 761 could protect spinal cord neurons from H(2)O(2)-induced toxicity. In primary spinal cord neurons isolated from embryonic day 14 rats, H(2)O(2) administration resulted in a significant decrease in the survival of spinal cord neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst 33342 nuclear staining showed that these cells die by apoptosis. Such neuronal death, however, was significantly reversed by EGb761 in a dose-dependent manner. Moreover, a marked increase in intracellular free radical generation was found after the H(2)O(2) administration which could be reversed almost completely by EGb761, indicating that inhibition of free radical generation is an important mechanism of the anti-apoptosis action of EGb761. Finally, treatment of cells with H(2)O(2) for 12 h reduced the expression of Bcl-2, an anti-apoptotic gene, by 70% but showed no effect on the level of Bax, a pro-apoptotic gene. EGb76 treatment, however, significantly reversed H(2)O(2)-induced reduction of Bcl-2 expression and inhibited Bax expression by 2.3-fold. Thus, our study provided evidence showing that the protective effect of EGb761 on spinal cord neuronal apoptosis after oxidative stress is mediated, at least in part, by its anti-oxidative action and regulation of apoptosis-related genes Bcl-2 and Bax.
Assuntos
Apoptose/genética , Peróxido de Hidrogênio/farmacologia , Neurônios , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/citologia , Animais , Apoptose/fisiologia , Células Cultivadas , Feminino , Ginkgo biloba , Humanos , Marcação In Situ das Extremidades Cortadas , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/patologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Amyloid beta protein (Abeta), the central constituent of senile plaques in Alzheimer's disease (AD), is known to exert toxic effects on cultured neurons. In the present study, the protective effect of panaxydol (PND) and panaxynol (PNN) on Abeta25-35-induced neuronal apoptosis and potential mechanisms were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN prior to 10 microM Abeta25-35 exposure resulted significantly in elevation of cell survival determined by MTT assay, TUNEL/Hoechst staining and western blot. Furthermore, a marked increase in calcium influx and intracellular free radical generation was found after Abeta25-35 exposure, which could be almost completely reversed by pretreatment of PND or PNN. PND and PNN could also alleviate Abeta25-35-induced early-stage neuronal degeneration. These results indicated that inhibition of calcium influx and free radical generation is a mechanism of the anti-apoptotic action of PND and PNN. Since Abeta plays critical roles in the pathogenesis of AD, these findings raise the possibility that PND and PNN reduce neurodegeneration in AD.
Assuntos
Córtex Cerebral/citologia , Di-Inos/farmacologia , Álcoois Graxos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides/toxicidade , Análise de Variância , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Regulação da Expressão Gênica/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas/métodos , Microscopia Confocal/métodos , Fragmentos de Peptídeos/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.
Assuntos
Proteína de Ligação a CREB/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Di-Inos/farmacologia , Álcoois Graxos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Animais , Aorta/citologia , Proteína de Ligação a CREB/biossíntese , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , DNA/efeitos dos fármacos , Di-Inos/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Álcoois Graxos/química , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Conformação Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Nerve growth factor (NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF- and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1 (Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.
RESUMO
Hyperphosphatemia is common in patients with chronic kidney disease and is increasingly associated with poor clinical outcomes. Current management of hyperphosphatemia with dietary restriction and oral phosphate binders often proves inadequate. Tenapanor, a minimally absorbed, small-molecule inhibitor of the sodium/hydrogen exchanger isoform 3 (NHE3), acts locally in the gastrointestinal tract to inhibit sodium absorption. Because tenapanor also reduces intestinal phosphate absorption, it may have potential as a therapy for hyperphosphatemia. We investigated the mechanism by which tenapanor reduces gastrointestinal phosphate uptake, using in vivo studies in rodents and translational experiments on human small intestinal stem cell-derived enteroid monolayers to model ion transport physiology. We found that tenapanor produces its effect by modulating tight junctions, which increases transepithelial electrical resistance (TEER) and reduces permeability to phosphate, reducing paracellular phosphate absorption. NHE3-deficient monolayers mimicked the phosphate phenotype of tenapanor treatment, and tenapanor did not affect TEER or phosphate flux in the absence of NHE3. Tenapanor also prevents active transcellular phosphate absorption compensation by decreasing the expression of NaPi2b, the major active intestinal phosphate transporter. In healthy human volunteers, tenapanor (15 mg, given twice daily for 4 days) increased stool phosphorus and decreased urinary phosphorus excretion. We determined that tenapanor reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux, an effect mediated exclusively via on-target NHE3 inhibition.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Isoquinolinas/farmacologia , Fosfatos/metabolismo , Trocador 3 de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonamidas/farmacologia , Adulto , Idoso , Animais , Sequência de Bases , Células Cultivadas , Impedância Elétrica , Epitélio/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal/efeitos dos fármacos , Íons/urina , Masculino , Camundongos , Pessoa de Meia-Idade , Potássio/metabolismo , Prótons , Ratos , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Proteínas de Junções Íntimas/metabolismo , Adulto JovemRESUMO
Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo.
Assuntos
Tecido Adiposo Marrom/metabolismo , Reprogramação Celular/genética , Adipogenia/efeitos dos fármacos , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/metabolismo , Animais , Bexaroteno , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , PPAR gama/metabolismo , Interferência de RNA , Receptor X Retinoide alfa/antagonistas & inibidores , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Receptor X Retinoide beta/antagonistas & inibidores , Receptor X Retinoide beta/genética , Receptor X Retinoide beta/metabolismo , Receptor X Retinoide gama/antagonistas & inibidores , Receptor X Retinoide gama/genética , Receptor X Retinoide gama/metabolismo , Tetra-Hidronaftalenos/farmacologia , Termogênese/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1/metabolismoRESUMO
We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/genética , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Camundongos , Serotonina/genética , Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.
Assuntos
Adipócitos Bege/metabolismo , Adipócitos Marrons/metabolismo , Gordura Subcutânea/metabolismo , Adipócitos Bege/efeitos dos fármacos , Adipócitos Marrons/efeitos dos fármacos , Animais , Axitinibe , Western Blotting , Orelha , Técnicas de Introdução de Genes , Teste de Tolerância a Glucose , Imidazóis/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Indazóis/farmacologia , Luciferases/genética , Medições Luminescentes , Masculino , Camundongos , Modelos Animais , Consumo de Oxigênio , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gordura Subcutânea/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1/genéticaRESUMO
Spinal cord neuronal culture is a useful system to study normal and abnormal functions of the spinal cord. For many bioassays, obtaining large quantities of highly purified spinal cord neurons is required. However, technical difficulties exist in obtaining these cells reliably and consistently. By comparing two dissociation methods, mechanical and enzymatic dissociations, we found that the enzymatic dissociation of embryonic day 14-15 spinal cords resulted in significantly higher cell yield than the mechanical dissociation (25.40 +/- 5.41 x 10(6) versus 3.43 +/- 0.52 x 10(6) cells per 12 embryos; n = 6/group; p < 0.01). Furthermore, cell viability was significantly higher after the enzymatic than the mechanical dissociation (83.40 +/- 3.08% versus 32.81 +/- 3.49%, n = 4/group; p < 0.01). In both methods, highly purified populations of primary neurons were obtained (mechanical: 85.17 +/- 2.84%; enzymatic: 87.67 +/- 2.52%; n = 3/group). Critical measures that affect culture outcomes include, but not limited to, the age of embryo, cell seeding density, dissociation time, and elimination of non-neuronal cells. Thus, the present study has identified the enzymatic dissociation method to be a preferred method for obtaining large quantity of highly-enriched embryonic spinal cord neurons.
Assuntos
Separação Celular/métodos , Enzimas/farmacologia , Mecânica , Neurônios/fisiologia , Medula Espinal , Fatores Etários , Animais , Contagem de Células/métodos , Células Cultivadas/efeitos dos fármacos , Embrião de Mamíferos , Feminino , Imunofluorescência , Gravidez , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Tubulina (Proteína)/metabolismoRESUMO
Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.
Assuntos
Alcinos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Álcoois Graxos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Animais , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Di-Inos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Células PC12 , Panax/química , RatosRESUMO
Stearic acid is a long-chain saturated fatty acid consisting of 18 carbon atoms without double bonds. In the present study, we reported the neuroprotective effects and mechanism of stearic acid on cortical or hippocampal slices insulted by oxygen-glucose deprivation, NMDA or hydrogen peroxide (H(2)O(2)) in vitro. Different types of models of brain slice injury in vitro were developed by 10 min of oxygen/glucose deprivation, 0.5 mM NMDA or 2 mM H(2)O(2), respectively. After 30 min of preincubation with stearic acid (3-30 microM), cortical or hippocampal slices were subjected to oxygen-glucose deprivation, NMDA or H(2)O(2). Then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride (TTC) method. Population spikes were recorded in randomly selected hippocampal slices. Stearic acid (3-30 microM) dose-dependently protected brain slices from oxygen-glucose deprivation, NMDA and H(2)O(2) insults. Its neuroprotective effect against H(2)O(2) insults can be completely blocked by wortmannin (inhibitor of PI3K) and partially blocked by H7 (inhibitor of PKC) or genistein (inhibitor of TPK). Treatment of cortical or hippocampal slices with 30 microM stearic acid resulted in a significant increase in PI3K activity at 5, 10, 30 and 60 min. These observations reveal that stearic acid can protect cortical or hippocampal slices against injury induced by oxygen-glucose deprivation, NMDA or H(2)O(2), and its neuroprotective effects are via phosphatidylinositol 3-kinase dependent mechanism.
Assuntos
Hipóxia Encefálica/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Ácidos Esteáricos/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Corantes/farmacologia , Relação Dose-Resposta a Droga , Glucose/deficiência , Glucose/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Hipóxia Encefálica/enzimologia , Hipóxia Encefálica/etiologia , Masculino , Técnicas de Cultura de Órgãos , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio/farmacologiaRESUMO
An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1mM SNP, an exogenous NO donor, exposure for 1h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 microM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nitroprussiato/toxicidade , Substâncias Protetoras/farmacologia , Alcinos/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Apoptose/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Di-Inos , Álcoois Graxos/farmacologia , Radicais Livres/metabolismo , Neurônios/patologia , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C. The chemically induced cardiomyocyte-like cells uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. 9C treatment of human fibroblasts resulted in a more open-chromatin conformation at key heart developmental genes, enabling their promoters and enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to chemically induced cardiomyocyte-like cells. This pharmacological approach to lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.
Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Miócitos Cardíacos/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Transdiferenciação Celular , Cromatina/química , Cromatina/metabolismo , Modelos Animais de Doenças , Fibroblastos/citologia , Coração/embriologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Cadeias Pesadas de Miosina/genética , Organogênese/genética , Conformação Proteica , Pele/citologia , Bibliotecas de Moléculas Pequenas/química , Transcriptoma , Transdução GenéticaRESUMO
Stem cell-based approaches to cardiac regeneration are increasingly viable strategies for treating heart failure. Generating abundant and functional autologous cells for transplantation in such a setting, however, remains a significant challenge. Here, we isolated a cell population with extensive proliferation capacity and restricted cardiovascular differentiation potentials during cardiac transdifferentiation of mouse fibroblasts. These induced expandable cardiovascular progenitor cells (ieCPCs) proliferated extensively for more than 18 passages in chemically defined conditions, with 10(5) starting fibroblasts robustly producing 10(16) ieCPCs. ieCPCs expressed cardiac signature genes and readily differentiated into functional cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) in vitro, even after long-term expansion. When transplanted into mouse hearts following myocardial infarction, ieCPCs spontaneously differentiated into CMs, ECs, and SMCs and improved cardiac function for up to 12 weeks after transplantation. Thus, ieCPCs are a powerful system to study cardiovascular specification and provide strategies for regenerative medicine in the heart.
Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Fibroblastos , Células-Tronco Pluripotentes Induzidas , Mioblastos Cardíacos , Infarto do Miocárdio , Transplante de Células-Tronco , Animais , Fibroblastos/metabolismo , Fibroblastos/transplante , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/transplante , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapiaRESUMO
Harmine is a natural compound possessing insulin-sensitizing effect in db/db diabetic mice. However its effect on adipose tissue browning is unknown. Here we reveal that harmine antagonizes high fat diet-induced adiposity. Harmine-treated mice gained less weight on a high fat diet and displayed increased energy expenditure and adipose tissue thermogenesis. In vitro, harmine potently induced the expression of thermogenic genes in both brown and white adipocytes, which was largely abolished by inhibition of RAC1/MEK/ERK pathway. Post-transcriptional modification analysis revealed that chromodomain helicase DNA binding protein 4 (CHD4) is a potential downstream target of harmine-mediated ERK activation. CHD4 directly binds the proximal promoter region of Ucp1, which is displaced upon treatment of harmine, thereby serving as a negative modulator of Ucp1. Thus, here we reveal a new application of harmine in combating obesity via this off-target effect in adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , DNA Helicases/metabolismo , Harmina/administração & dosagem , Neuropeptídeos/metabolismo , Termogênese , Proteínas rac1 de Ligação ao GTP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipócitos Marrons/citologia , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Animais , Células Cultivadas , DNA Helicases/genética , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Harmina/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Termogênese/efeitos dos fármacosRESUMO
Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.
Assuntos
Autofagia , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Proteína 5 Relacionada à Autofagia , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeos/farmacologia , Sirolimo/farmacologiaRESUMO
AIM: To investigation the anti-coxsackievirus B(3) (CVB(3m)) effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB(3m). METHODS: HeLa cells were infected with CVB(3m) and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A(600) was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A(600). RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Consistent results were got through these two methods. We also investigated the mechanism of its anti-CVB(3m) effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB(3m) attachment, entry, biosynthesis and assemble in cells. CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB(3m) in a dose-dependent manner and this effect exists through the process of CVB(3m) attachment, entry, biosynthesis and assemble in cells, suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.
Assuntos
Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Tricosantina/farmacologia , Acetatos , Células HeLa , Humanos , Extratos Vegetais/farmacologiaRESUMO
It was recently shown that mouse fibroblasts could be reprogrammed into cells of a cardiac fate by forced expression of multiple transcription factors and microRNAs. For ultimate application of such a reprogramming strategy for cell-based therapy or in vivo cardiac regeneration, reducing or eliminating the genetic manipulations by small molecules would be highly desirable. Here, we report the identification of a defined small-molecule cocktail that enables the highly efficient conversion of mouse fibroblasts into cardiac cells with only one transcription factor, Oct4, without any evidence of entrance into the pluripotent state. Small-molecule-induced cardiomyocytes spontaneously contract and exhibit a ventricular phenotype. Furthermore, these induced cardiomyocytes pass through a cardiac progenitor stage. This study lays the foundation for future pharmacological reprogramming approaches and provides a small-molecule condition for investigation of the mechanisms underlying the cardiac reprogramming process.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the expression of a few transcription factors has attracted enormous interest in biomedical research and the field of regenerative medicine. iPSCs nearly identically resemble embryonic stem cells (ESCs) and can give rise to all cell types in the body, and thus have opened new opportunities for personalized regenerative medicine and new ways of modeling human diseases. Although some studies have raised concerns about genomic stability and epigenetic memory in the resulting cells, better understanding and control of the reprogramming process should enable enhanced efficiency and higher fidelity in reprogramming. Therefore, small molecules regulating reprogramming mechanisms are valuable tools to probe the process of reprogramming and harness cell fate transitions for various applications.