RESUMO
The expression of apoptosis regulators (proapoptotic protein Bad and anti-apoptotic protein Bcl-2) was analyzed and Bcl-2/Bad ratio in the follicular apparatus of the rat ovary was determined on day 3 after hyperthermia (rectal temperature 43.5°C). Hyperthermia in the catabolic phase leads to different degrees of activation of the molecular "switches" of apoptosis in cells of ovarian follicular epithelium. This was seen from increased intensity of immunohistochemical staining for Bad protein against the background of more pronounced expression of Bcl-2 protein. On day 3 after exposure to hyperthermia, Bcl-2/Bad ratio increased, which reflects antiapoptotic protection of cells and conditions for blockade of mitochondrial pathway of apoptosis in the follicular apparatus of the ovaries during the acute period after hyperthermia.
Assuntos
Hipertermia Induzida/métodos , Folículo Ovariano/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína de Morte Celular Associada a bcl/genética , Animais , Apoptose/genética , Diestro/fisiologia , Feminino , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Folículo Ovariano/citologia , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/agonistas , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Animais , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/química , Dimerização , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Mobilização Lipídica , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Porosidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/agonistas , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismoRESUMO
Ghrelin functions as a neuroprotective agent and saves neurons from various insults include ischemic injury. However, it remains to be elucidated whether ghrelin protects neuronal cells against ischemic injury-induced excessive autophagy. Autophagy is required for the maintenance of neural stem cell homeostasis. However, regarding autophagic cell death, it is commonly assumed that excessive autophagy leads to self-elimination of mammalian cells. The purpose of this study was to investigate the potential neuroprotection effects of ghrelin from excessive autophagy in adult rat hippocampal neural stem cells (NSCs). Oxygen-Glucose Deprivation (OGD) strongly induces autophagy in adult rat hippocampal NSCs. Ghrelin treatment inhibited OGD-induced cell death of adult rat hippocampal NSCs assessed by cell-counting-kit-8 assay. Ghrelin also suppressed OGD-induced excessive autophagy activity. The protective effect of ghrelin was accompanied by an increased expression levels of Bcl-2, p-62 and decreased expression level of LC3-II, Beclin-1 by Western blot. Furthermore, ghrelin reduced autophagosome formation and number of GFP-LC3 transfected puncta. In conclusion, our data suggest that ghrelin protects adult rat hippocampal NSCs from excessive autophagy in experimental stroke (oxygen-glucose deprivation) model. Regulating autophagic activity may be a potential optimizing target for promoting adult rat hippocampal NSCs based therapy for stroke.
Assuntos
Autofagia , Grelina/metabolismo , Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neuroproteção , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteína Sequestossoma-1/agonistas , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Células-Tronco Adultas/ultraestrutura , Animais , Apoptose , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/metabolismo , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/patologia , Hipocampo/ultraestrutura , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Sequestossoma-1/metabolismoRESUMO
The aim of this study was to examine the comprehensive neuroprotective mechanism of ligustrazine, which is extracted from Ligusticum Chuanxiong Hort., against vascular dementia (VD) in rats and apoptosis in oxygen and glucose deprivation (OGD) PC12 cells. Rats were subjected to bilateral common carotid artery occlusion (BCCAO) surgery and administered ligustrazine intragastrically for 6 weeks. At the end of the experiments, the hippocampal biomarkers brain-derived neurotrophic factor (BDNF), monocyte chemotactic protein 1 (MCP-1), and homocysteine (Hcy) were examined. In experiments in vitro, OGD PC12 cells were treated with ligustrazine for 0.5, 1, 3, 6, 12, or 24 h. The cell-released biomarkers BDNF, MCP-1, and Hcy were examined. Microscopy, acridine orange-ethidium bromide (AO/EB) staining, and flow cytometry assays were performed to investigate apoptosis. Cleaved caspase-3, Bcl-2 associated X protein (Bax), and B cell lymphoma 2 (Bcl-2) expression was examined using Western blot assays. The results showed that biomarkers, including MCP-1 and Hcy, were significantly increased in both the in vivo and in vitro models, while the BDNF level was significantly decreased compared with the sham or vehicle models. Microscopy, AO/EB staining, and flow cytometry analysis showed that severe cell damage occurred in OGD PC12 cells, and apoptosis played a major role in this environment. Further Western blot studies showed that the apoptosis-related Bax/Bcl-2 protein ratio and cleaved caspase-3 were significantly increased in the experiment. However, ligustrazine profoundly suppressed the imbalance of these biomarkers, reduced cell damage, decreased the Bax/Bcl-2, and downregulated cleaved caspase-3. Pro- and anti-apoptotic biomarkers of multiple pathways including BDNF, MCP-1, and Hcy played a joint role in triggering the activation of the mitochondria-related Bax/Bcl-2 and caspase-3 apoptosis pathway in VD. Ligustrazine attenuated VD by comprehensively regulating BDNF, MCP-1, and Hcy and inactivating the Bax/Bcl-2 and caspase-3 apoptosis pathway. Our data provide novel insight into ligustrazine, which is a promising neuroprotective agent for VD disease treatment strategies. © IUBMB Life, 70(1):60-70, 2018.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/genética , Demência Vascular/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirazinas/farmacologia , Proteína X Associada a bcl-2/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Artéria Carótida Primitiva/cirurgia , Caspase 3/metabolismo , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Transtornos Cerebrovasculares , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Demência Vascular/genética , Demência Vascular/metabolismo , Demência Vascular/patologia , Regulação da Expressão Gênica , Glucose/deficiência , Glucose/farmacologia , Homocisteína/metabolismo , Ligusticum/química , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/isolamento & purificação , Células PC12 , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/isolamento & purificação , Ratos , Ratos Wistar , Transdução de Sinais , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismoRESUMO
We studied the effects of a melatonin-aluminum oxide-polymethylsiloxane complex (complex M) on the expression of apoptosis regulators Bcl-2 and Bad in the liver of homozygous db/db BKS.Cg-Dock7m+/+Leprdb/J mice with obesity and type 2 diabetes. Complex M or placebo was administered daily through the gastric tube during weeks 8-16 of life. In mice with type 2 diabetes mellitus receiving placebo, enhanced immunohistochemical reactions for proapoptotic Bad protein and weak response for anti-apoptotic Bcl-2 protein were observed. Administration of complex M shifted the ratio of apoptosis regulators: the area of Bcl-2 expression significantly increased and against the background of reduced Bad expression area. These findings attest to antiapoptotic effect of complex M in the liver on the model of type 2 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Melatonina/farmacologia , Obesidade/tratamento farmacológico , Substâncias Protetoras/farmacologia , Óxido de Alumínio/química , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Homozigoto , Fígado/metabolismo , Fígado/patologia , Melatonina/química , Camundongos , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Substâncias Protetoras/química , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Silicones/química , Proteína de Morte Celular Associada a bcl/antagonistas & inibidores , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Sperm cells can be damaged during the semen cryopreservation process, decreasing their fertilizing ability. Physical damage and oxidative stress may occur during the freeze-thawing process. Antioxidants such as the native antioxidant melatonin can potentially improve cryopreservation outcomes. In this study, we added melatonin to cryoprotectant to examine its effect on frozen-thawed human sperm. We found that adding 0.1mM melatonin to cryoprotectant significantly increased sperm viability (24.80 ± 0.46% vs. 20.97 ± 1.27%, P < 0.05) and membrane integrity (P < 0.05), and decreased intracellular reactive oxygen species and lipid peroxidation damage. Furthermore, mRNA levels of the transcription factor NF-E2-related factor-2 and its downstream genes were significantly increased. Resistance to oxidative stress was enhanced and expression of the antiapoptotic gene Bcl-2 was increased by inclusion of 0.1mM melatonin in the cryoprotectant. Moreover, 0.1mM melatonin upregulated the expression of heat shock protein 90 (HSP90), which confers resistance to stressors in frozen-thawed sperm. Results obtained upon addition of inhibitors of melatonin receptors (luzindole and 4-P-PDOT) and an HSP90 inhibitor (geldanamycin) in the cryoprotectant demonstrated that melatonin promoted HSP90 translation via the melatonin receptor MT1 and increased adenosine triphosphate levels, thus increasing the viability of thawed sperm.
Assuntos
Antioxidantes/farmacologia , Criopreservação/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/agonistas , Melatonina/farmacologia , Sêmen/efeitos dos fármacos , Trifosfato de Adenosina/biossíntese , Benzoquinonas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Receptor MT1 de Melatonina/antagonistas & inibidores , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Sêmen/citologia , Sêmen/metabolismo , Análise do Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacos , Tetra-Hidronaftalenos/farmacologia , Triptaminas/farmacologiaRESUMO
Myricitrin has multiple effects, including antagonism of platelet activating factor, regulation of blood sugar levels, oxidation resistance, protection of the liver and the relieving of ethylism. The present study evaluated how myricitrin weakens traumatic injury of the spinal cord (TISC), and exhibits antioxidant and antiinflammatory activities in a rat model. TISC model rats were injected intraperitoneally with 5, 10 or 30 mg/kg/day of myricitrin for 5 days. BassoBeattieBresnahan evaluation of locomotion and water content of spinal cord were used to analyze the effects of myricitrin on TISC. Myricitrin significantly inhibited the TISCinduced oxidative stress and inflammatory reactions. In addition, cyclooxygenase2 (COX2), transforming growth factor (TGF)ß1 and p53 were significantly reduced and Bcl2/Bax rate was significantly increased by treatment with myricitrin. The results of the current study suggested that the neuroprotective effect of myricitrin exhibits significant antioxidant and antiinflammatory activities, and a remarkable trauma protection activity in TISC rats through inhibition of COX2, TGFß1, p53 and elevation of Bcl2/Bax signaling pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Injeções Intraperitoneais , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Hair loss (alopecia) is a universal problem for numerous people in the world. The present study was conducted to investigate the effects of red ginseng oil (RGO) and its major components on hair re-growth using testosterone (TES)-induced delay of anagen entry in C57BL/6 mice and their mechanisms of action. Seven-week-old C57BL/6 mice were daily treated with TES for 1 h prior to topical application of 10% RGO, 1% linoleic acid (LA), 1% ß-sitosterol (SITOS), or 1% bicyclo(10.1.0)tridec-1-ene (BICYCLO) once a day for 28 days. Hair regenerative capacity was significantly restored by treatment of RGO and its major compounds in the TES-treated mice. Histological analysis showed that RGO along with LA and SITOS but not BICYCLO promoted hair growth through early inducing anagen phase that was delayed by TES in mice. Treatment of mice with RGO, LA, or SITOS up-regulated Wnt/ß-catenin and Shh/Gli pathways-mediated expression of genes such as ß-catenin, Lef-1, Sonic hedgehog, Smoothened, Gli-1, Cyclin D1, and Cyclin E in the TES-treated mice. In addition, RGO and its major components reduced the protein level of TGF-ß but enhanced the expression of anti-apoptotic protein Bcl-2. These results suggest that RGO is a potent novel therapeutic natural product for treatment of androgenic alopecia possibly through hair re-growth activity of its major components such as LA and SITOS.
Assuntos
Alopecia/tratamento farmacológico , Folículo Piloso/efeitos dos fármacos , Ácido Linoleico/farmacologia , Panax/química , Óleos de Plantas/farmacologia , Sitosteroides/farmacologia , Alopecia/induzido quimicamente , Alopecia/genética , Alopecia/patologia , Animais , Ciclinas/genética , Ciclinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óleos de Plantas/isolamento & purificação , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Testosterona/administração & dosagem , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Natural products are considered as an important source for the discovery of new drugs to treat aging-related degenerative diseases and liver injury. The present study profiled the chemical constituents of a methanol extract from Senna singueana bark using HPLC-PDA-ESI-MS/MS and 36 secondary metabolites were identified. Proanthocyanidins dominated the extract. Monomers, dimers, trimers of (epi)catechin, (epi)gallocatechin, (epi)guibourtinidol, (ent)cassiaflavan, and (epi)afzelechin represented the major constituents. The extract demonstrated notable antioxidant activities in vitro: In DPPH (EC50 of 20.8 µg/mL), FRAP (18.16 mM FeSO4/mg extract) assays, and total phenolic content amounted 474 mg gallic acid equivalent (GAE)/g extract determined with the Folin-Ciocalteu method. Also, in an in vivo model, the extract increased the survival rate of Caenorhabditis elegans worms pretreated with the pro-oxidant juglone from 43 to 64%, decreased intracellular ROS inside the wild-type nematodes by 47.90%, and induced nuclear translocation of the transcription factor DAF-16 in the transgenic strain TJ356. Additionally, the extract showed a remarkable hepatoprotective activity against d-galactosamine (d-GalN) induced hepatic injury in rats. It significantly reduced elevated AST (aspartate aminotransferase), and total bilirubin. Moreover, the extract induced a strong cytoplasmic Bcl-2 expression indicating suppression of apoptosis. In conclusion, the bark extract of S. sengueana represents an interesting candidate for further research in antioxidants and liver protection.
Assuntos
Antioxidantes/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Substâncias Protetoras/farmacologia , Senna/química , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/genética , Bilirrubina/sangue , Compostos de Bifenilo/antagonistas & inibidores , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Catecóis/química , Catecóis/isolamento & purificação , Catecóis/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Galactosamina/toxicidade , Masculino , Metanol/química , Naftoquinonas/antagonistas & inibidores , Naftoquinonas/farmacologia , Oxidantes/antagonistas & inibidores , Oxidantes/farmacologia , Fenóis/química , Fenóis/isolamento & purificação , Fenóis/farmacologia , Picratos/antagonistas & inibidores , Casca de Planta/química , Extratos Vegetais/química , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Proantocianidinas/farmacologia , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Solventes/químicaRESUMO
Aberrant modulation of mitochondrial dynamic network, which shifts the balance of fusion and fission towards fission, is involved in brain damage of various neurodegenerative diseases including Parkinson's disease, Huntington's disease and Alzheimer's disease. A recent research has shown that the inhibition of mitochondrial fission alleviates early brain injury after experimental subarachnoid hemorrhage, however, the underlying molecular mechanisms have remained to be elucidated. This study was undertaken to characterize the effects of the inhibition of dynamin-related protein-1 (Drp1, a dominator of mitochondrial fission) on blood-brain barrier (BBB) disruption and neuronal apoptosis following SAH and the potential mechanisms. The endovascular perforation model of SAH was performed in adult male Sprague Dawley rats. The results indicated Mdivi-1(a selective Drp1 inhibitor) reversed the morphologic changes of mitochondria and Drp1 translocation, reduced ROS levels, ameliorated the BBB disruption and brain edema remarkably, decreased the expression of MMP-9 and prevented degradation of tight junction proteins-occludin, claudin-5 and ZO-1. Mdivi-1 administration also inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB), leading to decreased expressions of TNF-É, IL-6 and IL-1ß. Moreover, Mdivi-1 treatment attenuated neuronal cell death and improved neurological outcome. To investigate the underlying mechanisms further, we determined that Mdivi-1 reduced p-PERK, p-eIF2α, CHOP, cleaved caspase-3 and Bax expression as well as increased Bcl-2 expression. Rotenone (a selective inhibitor of mitochondrial complexes I) abolished both the anti-BBB disruption and anti-apoptosis effects of Mdivi-1. In conclusion, these data implied that excessive mitochondrial fission might inhibit mitochondrial complex I to become a cause of oxidative stress in SAH, and the inhibition of Drp1 by Mdivi-1 attenuated early brain injury after SAH probably via the suppression of inflammation-related blood-brain barrier disruption and endoplasmic reticulum stress-based apoptosis.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Dinaminas/genética , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Quinazolinonas/farmacologia , Hemorragia Subaracnóidea/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Claudina-5/genética , Claudina-5/metabolismo , Dinaminas/antagonistas & inibidores , Dinaminas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Ocludina/genética , Ocludina/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Hemorragia Subaracnóidea/genética , Hemorragia Subaracnóidea/mortalidade , Hemorragia Subaracnóidea/patologia , Espaço Subaracnóideo/efeitos dos fármacos , Espaço Subaracnóideo/metabolismo , Espaço Subaracnóideo/patologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
It is well known that Panax ginseng (PG) has various pharmacological effects such as anti-aging and anti-inflammation. In a previous study, the authors identified that PG extract induced hair growth by means of a mechanism similar to that of minoxidil. In the present study, the inhibitory effect of PG extract on Dickkopf-1 (DKK-1)-induced catagen-like changes in hair follicles (HFs) was investigated in addition to the underlying mechanism of action. The effects of PG extract on cell proliferation, anti-apoptotic effect, and hair growth were observed using cultured outer root sheath (ORS) keratinocytes and human HFs with or without DKK-1 treatment. The PG extract significantly stimulated proliferation and inhibited apoptosis, respectively, in ORS keratinocytes. PG extract treatment affected the expression of apoptosis-related genes Bcl-2 and Bax. DKK-1 inhibited hair growth, and PG extract dramatically reversed the effect of DKK-1 on ex vivo human hair organ culture. PG extract antagonizes DKK-1-induced catagen-like changes, in part, through the regulation of apoptosis-related gene expression in HFs. These findings suggested that PG extract may reduce hair loss despite the presence of DKK-1, a strong catagen inducer via apoptosis.
Assuntos
Folículo Piloso/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Queratinócitos/efeitos dos fármacos , Panax/química , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Minoxidil/farmacologia , Extratos Vegetais/química , Raízes de Plantas/química , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vasodilatadores/farmacologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The genomic lesions that characterize acute lymphoblastic leukemia in childhood include recurrent translocations that result in the expression of fusion proteins that typically involve genes encoding tyrosine kinases, cytokine receptors, and transcription factors. These genetic rearrangements confer phenotypic hallmarks of malignant transformation, including unrestricted proliferation and a relative resistance to apoptosis. In this Minireview, we discuss the molecular mechanisms that link these fusions to the control of cell death. We examine how these fusion genes dysregulate the BCL-2 family of proteins, preventing activation of the apoptotic effectors, BAX and BAK, and promoting cell survival.
Assuntos
Modelos Biológicos , Fusão Oncogênica , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Animais , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Translocação GenéticaRESUMO
The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG), followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group (NC), COH implantation dysfunction model group (COH), low dosage of Bu-Shen-An-Tai recipe group (LOW), middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17ß-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis- related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of E2 and P4, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It's helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Implantação do Embrião/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Síndrome de Hiperestimulação Ovariana/prevenção & controle , Substâncias para o Controle da Reprodução/farmacologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Gonadotropina Coriônica/administração & dosagem , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Esquema de Medicação , Implantação do Embrião/fisiologia , Estradiol/sangue , Feminino , Absorção Gástrica/fisiologia , Gonadotropinas Equinas/administração & dosagem , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Cavalos , Camundongos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Síndrome de Hiperestimulação Ovariana/induzido quimicamente , Síndrome de Hiperestimulação Ovariana/genética , Síndrome de Hiperestimulação Ovariana/patologia , Indução da Ovulação/métodos , Gravidez , Progesterona/sangue , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Human umbilical cord mesenchymal stem cells (huMSCs) can treat primary ovarian insufficiency (POI) related to ovarian granulosa cell (OGC) apoptosis caused by cisplatin chemotherapy. Exosomes are a class of membranous vesicles with diameters of 30-200 nm that are constitutively released by eukaryotic cells. Exosomes mediate local cell-to-cell communication by transferring microRNAs and proteins. In the present study, we demonstrated the effects of exosomes derived from huMSCs (huMSC-EXOs) on a cisplatin-induced OGC model in vitro and discussed the preliminary mechanisms involved in these effects. We successfully extracted huMSC-EXOs from huMSC culture supernatant and observed the effective uptake of exosomes by cells with fluorescent staining. Using flow cytometry (with annexin-V/PI labelling), we found that huMSC-EXOs increased the number of living cells. Western blotting showed that the expression of Bcl-2 and caspase-3 were upregulated, whilst the expression of Bax, cleaved caspase-3 and cleaved PARP were downregulated to protect OGCs. These results suggest that huMSC-EXOs can be used to prevent and treat chemotherapy-induced OGC apoptosis in vitro. Therefore, this work provides insight and further evidence of stem cell function and indicates that huMSC-EXOs protect OGCs from cisplatin-induced injury in vitro.
Assuntos
Cisplatino/antagonistas & inibidores , Exossomos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Substâncias Protetoras/farmacologia , Animais , Anexina A5/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Cisplatino/farmacologia , Feminino , Citometria de Fluxo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Substâncias Protetoras/química , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Herein, a series of imidazo[4,5-f][1,10] phenanthroline derivatives RPIP (PIP = imidazo [4,5-f][1,10] phenanthroline, R = NO2, 1; CF3, 2; Cl, 3; OH, 4) have been synthesized in yields of 82.3-94.7% at 100 °C under the irradiation of microwave. MTT assay has been utilized to evaluate the inhibitory activity (IC50) of these compounds against the growth of various tumor cells, and the results revealed that these compounds, especially 1, exhibited excellent inhibitory activity against the growth of A549 cells with IC50 of 15.03 µM. Moreover, it's also confirmed that 1 can penetrate into the membrane of tumor cells and distribute in mitochondria when observed under microscopy, resulting apoptosis of tumor cells. The further studies showed that 1 can bind to bcl-2 G-quadruplex DNA, which demonstrated by the increase of melting point of bcl-2 G4 DNA in the presence of 1, as well as electronic titration and emission spectra. In a word, this kind of compound may develop as a potential apoptosis inducer in cancer chemotherapy via binding and stabilizing to the bcl-2 G-quadruplex DNA.
Assuntos
Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Imidazóis/síntese química , Mitocôndrias/efeitos dos fármacos , Fenantrolinas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Células A549 , Antineoplásicos/farmacologia , Transporte Biológico , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Imidazóis/farmacologia , Micro-Ondas , Mitocôndrias/metabolismo , Desnaturação de Ácido Nucleico , Fenantrolinas/farmacologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. Previous study indicates that calcium-sensing receptor (CaSR) activation contributes to neuron death in focal cerebral ischemia-reperfusion mice, however, its role in neuronal apoptosis after TBI is not well-established. Using a controlled cortical impact model in rats, the present study was designed to determine the effect of CaSR inhibitor NPS2390 upon neuronal apoptosis after TBI. Rats were randomly distributed into three groups undergoing the sham surgery or TBI procedure, and NPS2390 (1.5 mg/kg) was infused subcutaneously at 30 min and 120 min after TBI. All rats were sacrificed at 24 h after TBI. Our data indicated that NPS2390 significantly reduced the brain edema and improved the neurological function after TBI. In addition, NPS2390 decreased caspase-3 levels and the number of apoptotic neurons. Furthermore, NPS2390 up-regulated anti-apoptotic protein Bcl-2 expression and down-regulated pro-apoptotic protein Bax, and reduced subsequent release of cytochrome c into the cytosol. In summary, this study indicated that inhibition of CaSR by NPS2390 attenuates neuronal apoptosis after TBI, in part, through modulating intrinsic apoptotic pathway.
Assuntos
Adamantano/análogos & derivados , Edema Encefálico/tratamento farmacológico , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Quinoxalinas/farmacologia , Receptores de Detecção de Cálcio/antagonistas & inibidores , Adamantano/farmacologia , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/genética , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Caspase 3/genética , Caspase 3/metabolismo , Citocromos c/antagonistas & inibidores , Citocromos c/metabolismo , Regulação da Expressão Gênica , Infusões Subcutâneas , Masculino , Neurônios/metabolismo , Neurônios/patologia , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
We studied the effect of carbon monoxide (60-, 75-, and 90-min exposure) on the expression of antiapoptotic proteins (survivin and Bcl-2) in human blood lymphocytes in the presence of recombinant IL-2 in an apoptosis-inducing dose (0.1 ng/ml). Incubation of cells in atmosphere with carbon monoxide in the presence of recombinant IL-2 was accompanied by accumulation of Bcl-2 protein with simultaneous decrease of survivin content. It was concluded that carbon monoxide plays a role in the dysregulation of apoptosis of human blood lymphocytes Bcl-2 (i.e. CO inhibits the proapoptotic effect of recombinant IL-2).
Assuntos
Apoptose/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Proteínas Inibidoras de Apoptose/genética , Interleucina-2/farmacologia , Linfócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Apoptose/genética , Regulação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-2/antagonistas & inibidores , Linfócitos/citologia , Linfócitos/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , SurvivinaRESUMO
The protective effects of carnitine have been attributed to inhibition of apoptosis, alleviating oxidative stress and DNA repair mechanism by decreasing oxidative radicles. Carnitine also increases mitochondrial biogenesis via peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α). The role of carnitine in testicular PGC1α expression has not been documented. We hypothesised that the effects of carnitine as an antioxidant, inhibitor of apoptosis and controller of steroidogenesis in mouse testis may involve PGC1α as a regulator. The present study was designed to evaluate the localisation of PGC1α and the effects of carnitine treatment on the expression of PGC1α, Bcl2 and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) in mouse testis and serum testosterone concentrations. PGC1α was primarily immunolocalised to the Leydig cells and primary spermatocytes. Western blot analysis showed that carnitine (50mgkg-1 and 100mgkg-1 for 7 days) significantly increased PGC1α and Bcl2 expression in the testis in a dose-dependent manner. In addition, carnitine treatment significantly increased antioxidant enzyme (CAT, SOD and GPx) levels. The carnitine-induced changes in PGC1α in the testis were significantly correlated with changes in serum testosterone concentrations, as well as with changes in Bcl2 expression and antioxidant enzyme activity in the testis, as evaluated by electrophoresis. Therefore, the results of the present study suggest that carnitine treatment of mice increases PGC1α levels in the testis, which may, in turn, regulate steroidogenesis by increasing expression of Bcl2 and antioxidant enzymes.
Assuntos
Antioxidantes/uso terapêutico , Carnitina/uso terapêutico , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Infertilidade Masculina/prevenção & controle , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Testículo/efeitos dos fármacos , Fatores de Transcrição/agonistas , Animais , Antioxidantes/administração & dosagem , Apoptose/efeitos dos fármacos , Carnitina/administração & dosagem , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Infertilidade Masculina/sangue , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Injeções Intraperitoneais , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Camundongos Endogâmicos , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Espermatócitos/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatócitos/patologia , Testículo/metabolismo , Testículo/patologia , Testosterona/agonistas , Testosterona/sangue , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cardiac ankyrin repeat protein (CARP) is a nuclear transcriptional co-factor that has additional functions in the myoplasm as a component of the muscle sarcomere. Previous studies have demonstrated increased expression of CARP in cardiovascular diseases, however, its role in cardiomyocyte apoptosis is unclear and controversial. In the present study, we investigated possible roles of CARP in hypoxia/reoxygenation (H/R) -induced cardiomyocyte apoptosis and the underlying mechanisms. Neonatal mouse ventricular cardiomyocytes were isolated and infected with adenovirus encoding Flag-tagged CARP (Ad-CARP) and lentivirus encoding CARP targeted shRNA (sh-CARP), respectively. Cardiomyocyte apoptosis induced by exposure to H/R conditions was evaluated by TUNEL staining and western blot analysis of cleaved caspase-3. The results showed that H/R-induced apoptosis was significantly decreased in Ad-CARP cardiomyocytes and increased in sh-CARP cardiomyocytes, suggesting a protective anti-apoptosis role for CARP. Interestingly, over-expressed CARP was mainly distributed in the nucleus, consistent with its role in regulating transcriptional activity. qPCR analysis showed that Bcl-2 transcripts were significantly increased in Ad-CARP cardiomyocytes. ChIP and co-IP assays confirmed the binding of CARP to the Bcl-2 promoter through interaction with transcription factor GATA4. Collectively, our results suggest that CARP can protect against H/R induced cardiomyocyte apoptosis, possibly through increasing anti-apoptosis Bcl-2 gene expression.
Assuntos
Proteínas Musculares/genética , Isquemia Miocárdica/genética , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Traumatismo por Reperfusão/genética , Proteínas Repressoras/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
We have reported that hematopoietic system injury induced by total body irradiation (TBI) leads to generation of intracellular reactive oxygen species (ROS) and DNA damage, which are ameliorated by antioxidant agents. In the present study, we reported that administration of DIM, a potent antioxidant agent, not only protected mice against TBI-induced lethality, also ameliorated TBI-induced hematopoietic injury. The latter effect was probably attributable to DIM's inhibition of TBI-induced increases in ROS production in hematopoietic stem cells (HSCs) and the phosphorylation of histone H2AX (γ-H2AX). In particular, DIM led to significant improvements in bone marrow (BM) HSC frequency, hematopoietic progenitor cell (HPC) clonogenic function, and multilineage engraftment after transplantation. A downregulation of NADPH oxidase 4 (NOX4) and an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression were observed following DIM treatment. Notably, the anti-apoptotic potential of DIM was correlated with increased expression of the anti-apoptotic protein Bcl-2 and decreased expression of the pro-apoptotic protein Bax. These findings suggest that DIM attenuates TBI-induced hematopoietic injury through the inhibition of both oxidative stress in HSCs and hematopoietic cell apoptosis. Furthermore, we demonstrated that DIM protected BM hematopoietic cells against ionizing radiation and led to increased clonogenicity in vitro. Therefore, DIM has the potential to be used as an effective radioprotectant to ameliorate TBI-induced hematopoietic injury.