RESUMO
BACKGROUND & AIMS: The intestinal epithelium consists of EphB2-positive proliferative basal cryptic cells and EphrinB1-positive, postmitotic differentiated cells. We investigated the effects of Notch signaling on formation of the EphB2-EphrinB1 boundary using mouse and tissue culture models. METHODS: We created mice in which Mind bomb-1 (Mib1), an essential E3 ubiquitin ligase that activates Notch ligands, was inactivated specifically in the intestinal epithelia (Vil-Cre;Mib1(f/f)); Notch is, therefore, inactivated in this tissue. We also studied the effects of different inhibitors on intestinal epithelial cells (IEC-6) that express activated Notch. Tissues and cells were analyzed by immunohistochemical and immunoblot analyses. RESULTS: The intestinal epithelia of Vil-Cre;Mib1(f/f) mice had reduced numbers of EphrinB1-positive cells, compared with controls, but increases in EphB2-positive cells; beta-catenin was activated in these cells. These phenotypes were reversed by expression of a constitutively active form of Notch1. In the IEC-6 cells, Notch signaling activated the expression of EphrinB1 in an Hes1-independent manner, but down-regulated the expression of EphB2 through the GSK3beta-mediated inhibition of beta-catenin. CONCLUSIONS: Notch signaling regulates formation of the EphB2-EphrinB1 boundary in the mouse intestinal epithelium.
Assuntos
Efrina-B1/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Efrina-B2/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Receptor Notch1/genética , Fatores de Transcrição HES-1 , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Warm needle acupuncture (WNA) therapy combines the effects of acupuncture and heat produced by moxibustion. This therapy has been widely used in Korean traditional medicine to treat a number of health problems. We evaluated the analgesic effect of WNA treatment on formalin-induced pain behavior and c-Fos expression in the spinal cord of rats. Acupuncture and heat stimulation by moxibustion were performed at the Pungsi (GB31) acupoint. Needle insertion without heat stimulation (ACU) and heat stimulation without needle insertion (SWNA) were used as negative controls. WNA therapy was executed by burning 1.5 g of cylinder-shaped moxa on top of the needle that was inserted at the acupoint. We measured temperatures of two different locations on the needle using an automatic temperature-acquisition system. Needle temperatures were overwhelmingly dependent on the distance from moxa while burning and showed a maximum of 44.9 degrees C at the location 7 mm apart from the ground after ignition. WNA treatment was more effective than ACU or SWNA in alleviating pain during the late phase in the rat formalin test. WNA, ACU, and SWNA significantly reduced c-Fos expression in the superficial dorsal horn by 23.5, 28.3 and 19.4%, respectively.
Assuntos
Analgesia por Acupuntura/métodos , Pontos de Acupuntura , Temperatura Alta , Dor/prevenção & controle , Analgesia por Acupuntura/instrumentação , Análise de Variância , Animais , Comportamento Animal/fisiologia , Formaldeído , Imuno-Histoquímica , Masculino , Moxibustão/métodos , Dor/induzido quimicamente , Dor/fisiopatologia , Medição da Dor/métodos , Limiar da Dor , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Resultado do TratamentoRESUMO
The immature brain is affected profoundly by hypoxia-ischemia (HI) injury, which can lead to permanent neurologic sequelae in survivors. Neuronal degeneration after HI injury usually is achieved through apoptosis. Both CD95 and its natural ligand, CD95L, which are key molecules in the regulation of apoptosis, are constitutively expressed by neurons and astrocytes during embryonic and early postnatal stages. Further, CD95 or CD95L may have a functional relationship in glial cells and lead to apoptosis of these cells. The hippocampus, especially the CA1 area, is particularly susceptible to HI injury. We therefore investigated the temporal and spatial alterations in CD95 and CD95L expression in the CA1 area of 7-d-old rats after unilateral ligation of the carotid artery. Using immunohistochemistry and Western blotting, we showed that expression of CD95 and CD95L in the hippocampus peaked at 12 h and then decreased. In addition, we used terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling to demonstrate apoptosis among CD95- and CD95L-reactive cells. Our findings show that increases in the expression of CD95 and CD95L after HI injury may involve astrocytic apoptosis in the 7-d-old rat hippocampus, and these molecules may act as targets or inducers of cell death.