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Front Neurosci ; 14: 581915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33177984


Cerebral ischemia induces neuronal cell death and causes various kinds of brain dysfunction. Therefore, prevention of neuronal cell death is most essential for protection of the brain. On the other hand, it has been reported that epigenetics including DNA methylation plays a pivotal role in pathogenesis of some diseases such as cancer. Accumulating evidences indicate that aberrant DNA methylation is related to cell death. However, DNA methylation after cerebral ischemia has not been fully understood yet. The aim of this present study was to investigate the relationships between DNA methylation and neuronal cell death after cerebral ischemia. We examined DNA methylation under the ischemic condition by using transient middle cerebral artery occlusion and reperfusion (MCAO/R) model rats and N-methyl-D-aspartate (NMDA)-treated cortical neurons in primary culture. In this study, we demonstrated that DNA methylation increased in these neurons 24 h after MCAO/R and that DNA methylation, possibly through activation of DNA methyltransferases (DNMT) 3a, increased in such neurons immediately after NMDA treatment. Furthermore, NMDA-treated neurons were protected by treatment with a DNMT inhibitor that were accompanied by inhibition of DNA methylation. Our results showed that DNA methylation would be an initiation factor of neuronal cell death and that inhibition of such methylation could become an effective therapeutic strategy for stroke.

Mol Neurobiol ; 54(10): 7917-7927, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-27866373


Glycogen synthase kinase (GSK)-3ß, which is abundantly expressed in the central nervous system, regulates various cellular processes including gene expression, cell proliferation, and differentiation. However, involvement of GSK-3ß in cerebral ischemia-induced endogenous neurogenesis is not yet fully understood. Appropriate strategies to prevent ischemic cell damage and subsequent severe sequelae are needed. The purpose of the present study was to determine the relationship between pathophysiological alteration of the GSK-3ß signaling pathway and cerebral ischemia-induced endogenous neurogenesis in rats. Severe cerebral ischemia was produced by the injection of 700 microspheres into the right internal carotid artery of rats. We demonstrated that phosphorylation of GSK-3ß at its Ser9 and that of Akt was significantly enhanced on day 7 after the cerebral ischemia, as was the number of NeuroD-positive cells. Treatment with a phosphatidylinositol 3-kinase (PI3-K) inhibitor decreased the cerebral ischemia-induced phosphorylation of Akt and that of GSK-3ß at its Ser9. In addition, as the protein levels of insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) were decreased, they might not have been essential for activation of the PI3-K/Akt/GSK-3ß pathway after severe cerebral ischemia. Although it remains to be determined what factors activate this pathway, our results suggest that PI3K/Akt-dependent GSK-3ß signaling and subsequent expression of NeuroD were involved in the neurogenesis elicited by cerebral ischemia.

Isquemia Encefálica/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurogênese/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proliferação de Células/fisiologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Ratos Wistar , Transdução de Sinais/fisiologia
Transpl Immunol ; 33(2): 140-5, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26209355


BACKGROUND: Xenotransplantation is considered to be one of the most attractive strategies for overcoming the worldwide shortage of organs. However, many obstructions need to be overcome before it will achieve clinical use in patients. One such obstacle is the development of an effective immunosuppressive strategy. We previously reported that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of progenitor and immature myeloid cells, suppress xenogenic CTL-mediated cytotoxicity. Because of their heterogeneous nature, MDSC can function via several suppressive mechanisms that disrupt both innate and adaptive immunity. Since macrophages play a pivotal role in the rejection of a xenograft, in this study, we evaluated the suppressive effects of MDSC against macrophage-mediated xenogenic rejection. MATERIALS AND METHODS: To evaluate the effect of monocyte-derived MDSCs on xenogenic immune reactions, a CFSE(carboxyfluorescein diacetate, succinimidyl ester)assay was employed to assess cytotoxicity. RESULTS: While, in the absence of activation, primed MDSCs had no detectable effect on macrophage-induced cytotoxicity against SEC cells, LPS-activated MDSCs were found to significantly suppress xenogenic cytotoxicity. A CFSE cytotoxicity assay revealed that MDSCs significantly suppressed macrophage-induced cytotoxicity. Furthermore, an indoleamine 2,3 dioxygenase (IDO) inhibitor, 1-methyl tryptophan (1-MT), abolished the MDSC-induced suppression of macrophage-mediated xeno-rejection, indicating that MDSCs may suppress macrophage-mediated cytotoxicity in an IDO-dependent manner. CONCLUSION: These findings indicate that MDSCs have great potential for immunosuppressing macrophage-mediated xeno-rejection.

Rejeição de Enxerto/prevenção & controle , Macrófagos/imunologia , Células Mieloides/imunologia , Transplante Heterólogo , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Humanos , Imunossupressão , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Óxido Nítrico/metabolismo , Triptofano/análogos & derivados , Triptofano/farmacologia
Biosci Biotechnol Biochem ; 79(10): 1719-24, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26096293


Quorum sensing is a cell-to-cell communication mechanism, which is responsible for regulating a number of bacterial virulence factors and biofilm maturation and therefore plays an important role for establishing wound infection. Quorum-sensing signals may induce inflammation and predispose wounds to infection by Pseudomonas aeruginosa; however, the interaction has not been well investigated. We examined the effects of the P. aeruginosa las quorum-sensing signal, N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), on matrix metalloproteinase (MMP) 9 expression in Rat-1 fibroblasts. 3OC12-HSL upregulated the expression of the MMP9 gene bearing an activator protein-1 (AP-1) binding site in the promoter region. We further investigated the mechanism underlying this effect. c-Fos gene expression increased rapidly after exposure to 3OC12-HSL, and nuclear translocation of c-Fos protein was observed; both effects were reduced by pretreatment with an AP-1 inhibitor. These results suggest that 3OC12-HSL can alter MMP9 gene expression in fibroblasts via the AP-1 signaling pathway.

4-Butirolactona/análogos & derivados , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Homosserina/análogos & derivados , Metaloproteinase 9 da Matriz/genética , Pseudomonas aeruginosa/química , Fator de Transcrição AP-1/genética , 4-Butirolactona/farmacologia , Abietanos/farmacologia , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Homosserina/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Ratos , Transdução de Sinais , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo
Transpl Immunol ; 32(2): 109-15, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25559170


BACKGROUND: Xenotransplantation is an appealing alternative to human allotransplantation because of a worldwide shortage of organs. One of the obstacles for xenografts is cellular rejection by the innate immune system, comprised of NK cells, monocytes, and macrophages. In this study the inhibitory function of HLA-G1, a MHC Ib molecule, on macrophage-mediated cytotoxicity was examined. Furthermore, this study also evaluates the suppressive effect of cytokine production by macrophages. METHODS: The expression of inhibitory receptors that interact with HLA-G1, immunoglobulin-like transcript 2 (ILT2), ILT4 and KIR2DL4 (CD158d) on in vitro generated macrophages were examined by flow cytometry. Complementary DNA (cDNA) of HLA-G1, HLA-E and human ß2-microglobulin (hß2m) were prepared and transfected into swine endothelial cells (SECs). The expression of the transgenic genes was evaluated by flow cytometry, and macrophage-mediated SEC cytolysis was assessed using the macrophages. RESULTS: In vitro generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on SECs indicated significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. Furthermore, the results on real time PCR and ELISA revealed that transgenic HLA-G1 induces the anti-inflammatory cytokines, such as IL-10 and TGF-ß, and suppresses iNOS mRNA expression, indicating that transgenic HLA-G1 has suppressive effects in a broad range of transplant rejection. CONCLUSION: These results indicate that generating HLA-G1 transgenic pigs can protect porcine grafts from macrophage-mediated cytotoxicity.

Células Endoteliais/imunologia , Expressão Gênica , Antígenos HLA-G , Imunidade Celular/genética , Macrófagos/imunologia , Animais , Animais Geneticamente Modificados , Técnicas de Cocultura , Células Endoteliais/patologia , Feminino , Antígenos HLA-G/genética , Antígenos HLA-G/imunologia , Humanos , Macrófagos/patologia , Masculino , Suínos