RESUMO
Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.
Assuntos
Antraz , Bacillus anthracis , Leucemia Eritroblástica Aguda , Trombocitopenia , Animais , Humanos , Camundongos , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Fator 1 de Resposta a Butirato/metabolismo , Diferenciação Celular , Trombocitopenia/induzido quimicamente , Trombocitopenia/genéticaRESUMO
Established from the calvaria of newborn macrophage colony-stimulating factor (M-CSF)-deficient mice, OP9 is a stromal cell line that used as a feeder layer to support the in vitro differentiation of pluripotent stem cells into various hematopoietic lineage cells, including granulocytes, erythrocytes, lymphocytes, and megakaryocytes. However, as a primary culture cell line, OP9 can be used as stromal cells for only 1 month. Therefore, to obtain functional OP9 cells, numerous M-CSF-deficient newborn mice must be sacrificed. These limitations in some ways restrict the application of OP9 cells in longterm and largescale experiments. In this study, we used human papillomavirus 16 E6 and E7 genes to generate immortalized OP9 stromal cells, designated I-OP9 cells, and then tested their ability to support the megakaryocytic differentiation of pluripotent stem cells in vitro. I-OP9 cells have similar morphology and properties as do parental OP9 cells, and, as expected, have an extended lifespan and can support megakaryocytic differentiation. Our data suggest that the method used in this study, including establishing I-OP9 cells, enables the possibility to enlarge and lengthen the scale of the experiment and, more critically, provides a humanistic approach for preparing stromal cells that support the hematopoietic differentiation of pluripotent stem cells in vitro.
Assuntos
Diferenciação Celular , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Megacariócitos/citologia , Células-Tronco Embrionárias Murinas/citologia , Células Estromais/citologia , Animais , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Embrião de Mamíferos/metabolismo , Citometria de Fluxo , Imunofluorescência , Hematopoese , Humanos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismoRESUMO
BACKGROUND: Solar ultraviolet (UV) radiation causes various deleterious effects, and UV blockage is recommended for avoiding sunburn. Nanosized titanium dioxide and zinc oxide offer effective protection and enhance cosmetic appearance but entail health concerns regarding their photocatalytic activity, which generates reactive oxygen species. These concerns are absent in nanodiamonds (NDs). Among the UV wavelengths in sunlight, UVB irradiation primarily threatens human health. RESULTS: The efficacy and safety of NDs in UVB protection were evaluated using cell cultures and mouse models. We determined that 2 mg/cm(2) of NDs efficiently reduced over 95% of UVB radiation. Direct UVB exposure caused cell death of cultured keratinocyte, fibroblasts and skin damage in mice. By contrast, ND-shielding significantly protected the aforementioned pathogenic alterations in both cell cultures and mouse models. CONCLUSIONS: NDs are feasible and safe materials for preventing UVB-induced skin damage.
Assuntos
Nanodiamantes , Protetores contra Radiação/farmacologia , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Animais , Células Cultivadas , Dermatite/etiologia , Dermatite/prevenção & controle , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Hiperplasia/tratamento farmacológico , Hiperplasia/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Leucócitos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Protetores contra Radiação/química , Radiodermite/etiologia , Radiodermite/prevenção & controle , Pele/patologia , Titânio/farmacologia , Raios Ultravioleta/efeitos adversos , Óxido de Zinco/farmacologiaRESUMO
Excessive reactive oxygen species is the major component of a harsh microenvironment after ischemia/reperfusion injury in human tissues. Combined treatment of N-acetyl-L-cysteine (NAC) and L-ascorbic acid 2-phosphate (AAP) promoted the growth of human mesenchymal stem cells (hMSCs) and suppressed oxidative stress-induced cell death by enhancing mitochondrial integrity and function in vitro. In this study, we aimed to determine whether NAC and AAP (termed MCA) could enhance the therapeutic potential of hMSCs. We established a coculture system consisting of MCA-treated and H2O2-treated hMSCs and investigated the role of tunneling nanotubes (TNTs) in the exchange of mitochondria between the 2 cell populations. The consequences of mitochondria exchange were assessed by fluorescence confocal microscopy and flow cytometry. The results showed that MCA could increase the mitochondrial mass, respiratory capacity, and numbers of TNTs in hMSCs. The "energized" mitochondria were transferred to the injured hMSCs via TNTs, the oxidative stress was decreased, and the mitochondrial membrane potential of the H2O2-treated hMSCs was stabilized. The transfer of mitochondria decreased the expression of S616-phosphorylated dynamin-related protein 1, a protein that dictates the fragmentation/fission of mitochondria. Concurrently, MCA also enhanced mitophagy in the coculture system, implicating that damaged mitochondria were eliminated in order to maintain cell physiology.
Assuntos
Antioxidantes/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Dengue virus (DENV) infection can cause severe, life-threatening events, and no specific treatments of DENV infection are currently approved. Although thrombocytopenia is frequently observed in dengue patients, its pathogenesis is still not fully understood. Previous studies have suggested that DENV-induced thrombocytopenia occurs through viral-replication-mediated megakaryopoiesis inhibition in the bone marrow; however, the exact mechanism for megakaryopoiesis suppression remains elusive. In this study, a reductionist approach was applied, in which C57B/6J mice were inoculated with recombinant DENV-envelope protein domain III (DENV-EIII) instead of the full viral particle. Our results demonstrated that DENV-EIII-suppressed megakaryopoiesis is similar to those observed with DENV infection. Furthermore, in agreement with our in vivo analyses, DENV-EIII sufficiently suppressed the megakaryopoiesis of progenitor cells from murine bone marrow and human cord blood in vitro. Additional analyses suggested that autophagy impairment and apoptosis are involved in DENV-EIII-mediated suppression of megakaryopoiesis. These data suggest that, even without viral replication, the binding of DENV-EIII to the cell surface is sufficient to suppress megakaryopoiesis.
Assuntos
Vírus da Dengue/metabolismo , Dengue/fisiopatologia , Trombopoese , Proteínas do Envelope Viral/metabolismo , Animais , Autofagia , Linhagem Celular , Dengue/virologia , Vírus da Dengue/química , Vírus da Dengue/genética , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Envelope Viral/genéticaRESUMO
Lethal toxin (LT) that composed by protective antigen and lethal factor (LF) is the major virulence factor of Bacillus anthracis. The treatments of LT in animals could reproduce most manifestations of B. anthracis infections that greatly improves our knowledge in LT-mediated pathogenesis and facilitates anthrax-related researches without having to directly contact the hazardous bacterium B. anthracis. The recombinant protein of LF (rLF), however, still lacks a simple purification method. Herein, we developed single-step nickel affinity purification of rLF with yield up to 3mg/l. By fusion to the leader sequence of outer membrane protein OmpA, rLF could easily be purified from the periplasm of Escherichia coli. To investigate whether the rLT is functional in our system, both wild type rLF and the catalytic mutant rLF that contains a single amino acid substitution at zinc-binding site (LF(E687A)), were subjected to macrophage cytotoxicity analysis. Our data showed that the rLT is fully functional, while the LF(E687A) fail to induce cell death of tested macrophage cells. These findings suggested that the purification protocol herein is a user-friendly method that allows researchers to obtain the functional rLF by single-step purification.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/metabolismo , Toxinas Bacterianas/isolamento & purificação , Escherichia coli/metabolismo , Periplasma/química , Engenharia de Proteínas/métodos , Extração em Fase Sólida/métodos , Animais , Antígenos de Bactérias/farmacologia , Bacillus anthracis/genética , Toxinas Bacterianas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl2)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl2-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl2 exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.
Assuntos
Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Fitoquímicos/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Hipóxia Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cobalto/toxicidade , Flavonoides/antagonistas & inibidores , Flavonóis , Sequestradores de Radicais Livres/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/antagonistas & inibidores , Células PC12 , Compostos Fitoquímicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Ratos , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Emerging life threatening pathogens such as severe acute aspiratory syndrome-coronavirus (SARS-CoV), avian-origin influenzas H7N9, and the Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a high case-fatality rate and psychological effects on society and the economy. Therefore, a simple, rapid, and safe method to investigate a therapeutic approach against these pathogens is required. In this study, a simple, quick, and safe cell adhesion inhibition assay was developed to determine the potential cellular binding site on the SARS-CoV spike protein. Various synthetic peptides covering the potential binding site helped to minimize further the binding motif to 10-25 residues. Following analyses, 2 peptides spanning the 436-445 and 437-461 amino acids of the spike protein were identified as peptide inhibitor or peptide vaccine candidates against SARS-CoV.
Assuntos
Adesão Celular , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral , Animais , Antivirais/isolamento & purificação , Sítios de Ligação , Linhagem Celular , Humanos , Peptídeos/isolamento & purificação , Ligação ProteicaRESUMO
SCOPE: Curcumin has been demonstrated as having numerous desirable characteristics, such as antioxidant, anti-inflammatory, and antiatherogenic activities. We report the hypocholesterolemic effect and molecular mechanism of curcumin. METHODS AND RESULTS: We found that curcumin enhanced LDL receptor (LDLR) level on the cell surface, as well as LDLR activity; however, LDLR transcription and mRNA stability were not affected. Furthermore, we found that proprotein convertase subtilisin/kexin type 9 (PCSK9) gene was downregulated at the transcriptional level by curcumin, leading to an increase in LDL uptake in HepG2 cells. The curcumin-responsive element of the PCSK9 promoter, a binding site for hepatocyte nuclear factor 1α (HNF-1α), was also identified. We demonstrated that curcumin reduced the nuclear abundance of hepatocyte nuclear factor 1α, resulting in its attenuated interaction with the PCSK9 promoter and leading to a downregulation of PCSK9 expression. Finally, we showed that curcumin decreased the statin-induced PCSK9 expression and potentially synergized with statin administration. CONCLUSION: Current results indicate that curcumin suppression of PCSK9 expression is associated with increases in cell-surface LDLR and LDLR activity in hepatic cells and it acts in a molecular mechanism that is distinct from the statins. Curcumin exhibits hypolipidemic activity and may serve as a useful supplement to statin treatment for hypercholesterolemia.
Assuntos
LDL-Colesterol/metabolismo , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Curcumina/química , Sinergismo Farmacológico , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Serina Endopeptidases/genética , Transcrição GênicaRESUMO
Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34(+)-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax.
Assuntos
Antraz/microbiologia , Antraz/patologia , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Progressão da Doença , Eritropoese/efeitos dos fármacos , Anemia/complicações , Anemia/patologia , Animais , Antraz/complicações , Apoptose/efeitos dos fármacos , Biocatálise/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Células Eritroides/patologia , Eritropoetina/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Análise de SobrevidaRESUMO
Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34(+) cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.