RESUMO
In birds, primordial germ cells (PGCs) use the bloodstream to travel to a specific region, where the cells undergo extravasation followed by intrastromal migration to the gonadal crest for further colonization. Currently, DDX4, SSEA1, and Oct4 are used to identify germ cells. Other germline cell-associated molecules are N-cadherin, GnRHR, and 3ß hydroxysteroid dehydrogenase (3ßHSD), which have been used in mice and birds during gonadal development; however, its role in early gonadogenesis in birds is poorly described. This study aimed to evaluate the differential immunodetection of N-cadherin binding molecule, Oct4 pluripotency protein, GnRHR receptor, and 3ßHSD enzyme in Columba livia embryos during migration colonization of PGCs in the gonadal crest and early gonadogenesis. These markers were revealed by immunohistochemistry in histological preparations of C. livia corresponding to stages (S)15 to S40. Immunodetection of N-cadherin, Oct4, GnRHR, and 3ßHSD in the germ line of C. livia allowed the identification of PGCs in the yolk sac membrane at the level of the splanchnic mesoderm during migration to the genital crest and its colonization. In the same way, it was possible to characterize and localize PGCs during early gonadogenesis. This study in C. livia demonstrates that Oct4, N-cadherin, GNRHR, and 3ßHSD are immunodetected in PGCs and could be used as potential germline cell markers during cell migration out of blood vessels, colonization in the genital crest, and early gonadogenesis. Furthermore, this study could be used as a novel general model to understand the early gonadogenesis in altricial species.
Assuntos
Columbidae , Columbiformes , Animais , Camundongos , Células Germinativas/metabolismo , Diferenciação Celular , Movimento Celular , Caderinas/metabolismoRESUMO
The gular gland is a skin gland located in the suprasternal region of adult males of some bat families. Knowledge of the morphology and functional aspects of these gland types is often limited. This study aimed to describe the structure and composition of the gular glands of three molossid species (Eumops patagonicus, Molossus fluminensis and Molossus molossus) with respect to their reproductive activity and to define the mechanism involved in secretion release. Different histological, histochemical and immunohistochemical techniques were used to achieve these goals. The results revealed that the size and composition of this gland are variable and are mostly related to the lipid content during the reproductive season. The results also documented, for the first time, the occurrence of mechanoreceptors associated with the surface of the glandular duct by detecting an S100 protein, indicating that an external stimulus activates secretion. Previous studies on other species have classified the gland using obsolete criteria; hence, we adopted a new classification of adenomeres in this study. Moreover, we investigated the gland secretion mechanism previously proposed. This study defines the implications of this gland in the reproduction of this species. Our preliminary interpretation of the function of the gular gland is that it is a cutaneous exocrine gland activated by mechanoreceptors involved in the reproductive behaviour of the Molossidae family.
Assuntos
Quirópteros , Humanos , Masculino , Animais , Quirópteros/anatomia & histologia , Reprodução/fisiologia , América do Sul , Estações do AnoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Nectandra angustifolia belongs to the Lauraceae family and it is widely known in phytomedicine by local inhabitants of South America against various maladies. It is popularly used for the treatment of different types of inflammatory processes, like rheumatism, arthritis and its associated pain. AIM OF THE STUDY: To characterize the phytochemicals in an ethanolic extract of Nectandra angustifolia and to evaluate the total antioxidant content and its anti-inflammatory effect with multiparametric analyses through in vitro assays and an in vivo model. METHODS: Leaves and stems of Nectandra angustifolia were air-dried and an ethanolic extract (NaE) was further obtained. Total phenolic, flavonoid and tannin content were determined and the antioxidant activity was addressed by DPPH and FRAP assays. NaE was first analyzed by HPLC and then two tests were carried out as screening assays for anti-inflammatory activities: red blood cell membrane stabilization and protein denaturation. The non-cytotoxic concentration of NaE was determined for in vitro biological assays using RAW 264.7 (murine macrophages) cell cultures through cell counting with Trypan-blue and XTT assay. Subsequently, the cell cycle of RAW 264.7 cells exposed for 24 h to NaE was analyzed. Additionally, the anti-inflammatory capacity of NaE was evaluated by RT-qPCR of pro-inflammatory cytokines. Furthermore, NF-κB translocation was observed by confocal microscopy at different times. Finally, formalin-induced mice paw inflammation was used as an in vivo model. RESULTS: The chromatographic profile of NaE showed peaks compatible with flavonoids content. NaE exhibited better membrane stabilization effect on HRBC and protection of BSA denaturation than the standard drug (diclofenac). NaE diminished mRNA levels of pro-inflammatory cytokines when added 1-h prior LPS stimulation. Moreover, NaE prevented the translocation of NF-κB to the nucleus and in formalin-induced mice paw inflammation, reduced the edema and the stimulus of inflammatory phase. CONCLUSION: This study shows for the first time, that Nectandra angustifolia ethanolic extract has a high content of flavonoids and that possess antioxidant and anti-inflammatory biological properties as demonstrated by multiparametric analyses from in vitro assays and an in vivo model of inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Lauraceae/química , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/química , Antioxidantes/química , Antioxidantes/farmacologia , Comportamento Animal/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citocinas/antagonistas & inibidores , Modelos Animais de Doenças , Edema/induzido quimicamente , Edema/tratamento farmacológico , Eritrócitos/efeitos dos fármacos , Etanol/química , Formaldeído/toxicidade , Humanos , Inflamação/induzido quimicamente , Masculino , Camundongos , Compostos Fitoquímicos/análise , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Estabilidade Proteica/efeitos dos fármacos , Células RAW 264.7 , Fator de Transcrição RelA/metabolismoRESUMO
Kidney cancer accounts for 2.5% of all cancers, with an annual global incidence of almost 300,000 cases leading to 111,000 deaths. Approximately 85% of kidney tumors are renal cell carcinoma (RCC) and their major histologic subtype is clear cell renal cell carcinoma (ccRCC). Although new therapeutic treatments are being designed and applied based on the combination of tyrosine kinase inhibitors and immunotherapy, no major impact on the mortality has been reported so far. MRP4 is a pump efflux that transporters multiple endogenous and exogenous substances. Recently it has been associated with tumoral persistence and cell proliferation in several types of cancer including pancreas, lung, ovary, colon, ostesarcoma, etc. Herein, we demonstrate for the first time, that MRP4 is overexpressed in ccRCC tumors, compared to control renal tissues. In addition, using cell culture models, we observed that MRP4 pharmacological inhibition produces an imbalance in cAMP metabolism, induces cell arrest, changes in lipid composition, increase in cytoplasmic lipid droplets and finally apoptosis. These data provide solid evidence for the future evaluation of MRP4 as a possible new therapeutic target in ccRCC.
Assuntos
Carcinoma de Células Renais/genética , Proliferação de Células/genética , Neoplasias Renais/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Apoptose/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , AMP Cíclico/genética , Células HCT116 , Humanos , Rim/metabolismo , Neoplasias Renais/metabolismoRESUMO
Acute kidney injury (AKI) is a frequent complication of sepsis, with a high mortality. Hallmarks of septic-AKI include inflammation, endothelial injury, and tissue hypoxia. Therefore, it would be of interest to develop therapeutic approaches for improving the microvascular damage in septic-AKI. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone. Thus, the aim of this study was to evaluate the protective effects of EPO on microvascular injury in a murine model of endotoxemic AKI. Male Balb/c mice were divided into four groups: control, LPS (8 mg/kg, ip.), EPO (3000 IU / kg, sc.) and LPS + EPO. A time course study (0-48 h) was designed. Experiments include, among others, immunohistochemistry and Western blottings of hypoxia-inducible transcription factor (HIF-1α), erythropoietin receptor (EPO-R), vascular endothelial growth factor system (VEGF/VEGFR-2), platelet and endothelial adhesion molecule-1 (PeCAM-1), inducible nitric oxide synthase (iNOS) and phosphorylated nuclear factor kappa B p65 (NF-κB). Data showed that EPO attenuates renal microvascular damage during septic-AKI progression through a) the decrease of HIF-1 alpha, iNOS, and NF-κB and b) the enhancement of EPO-R, PeCAM-1, VEGF, and VEGFR-2 expression. In summary, EPO renoprotection involves the attenuation of septic-induced renal hypoxia and inflammation as well as ameliorates the endotoxemic microvascular injury.
Assuntos
Injúria Renal Aguda/prevenção & controle , Eritropoetina/farmacologia , Microvasos/efeitos dos fármacos , Sepse/tratamento farmacológico , Injúria Renal Aguda/etiologia , Animais , Western Blotting , Modelos Animais de Doenças , Progressão da Doença , Endotoxemia/complicações , Endotoxemia/tratamento farmacológico , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microvasos/patologia , Sepse/complicações , Fatores de TempoRESUMO
Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinomas. There is great interest to know the molecular basis of the tumor biology of ccRCC that might contribute to a better understanding of the aggressive biological behavior of this cancer and to identify early biomarkers of disease. This study describes the relationship among proliferation, survival, and apoptosis with the expression of key molecules related to tumoral hypoxia (hypoxia-inducible factor (HIF)-1α, erythropoietin (EPO), vascular endothelial growth factor (VEGF)), their receptors (EPO-R, VEGFR-2), and stearoyl desaturase-1 (SCD-1) in early stages of ccRCC. Tissue samples were obtained at the Urology Unit of the J.R. Vidal Hospital (Corrientes, Argentina), from patients who underwent radical nephrectomy for renal cancer between 2011 and 2014. Four experimental groups according to pathological stage and nuclear grade were organized: T1G1 (n = 6), T2G1 (n = 4), T1G2 (n = 7), and T2G2 (n = 7). The expression of HIF-1α, EPO, EPO-R, VEGF, VEGFR-2, Bcl-xL, and SCD-1 were evaluated by immunohistochemistry, Western blotting, and/or RT-PCR. Apoptosis was assessed by the TUNEL in situ assay, and tumor proliferation was determined by Ki-67 immunohistochemistry. Data revealed that HIF-1α, EPO, EPO-R, VEGF, and VEGF-R2 were overexpressed in most samples. The T1G1 group showed the highest EPO levels, approximately 200 % compared with distal renal tissue. Bcl-xL overexpression was concomitant with the enhancement of proliferative indexes. SCD-1 expression increased with the tumor size and nuclear grade. Moreover, the direct correlations observed between SCD-1/HIF-1α and SCD-1/Ki-67 increments suggest a link among these molecules, which would determine tumor progression in early stages of ccRCC. Our results demonstrate the relationship among proliferation, survival, and apoptosis with the expression of key molecules related to tumoral hypoxia (HIF-1α, EPO, VEGF), their receptors (EPO-R, VEGFR-2), and SCD-1 in early stages of ccRCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Eritropoetina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/patologia , Receptores da Eritropoetina/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Western Blotting , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/cirurgia , Proliferação de Células , Eritropoetina/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas Imunoenzimáticas , Neoplasias Renais/metabolismo , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Eritropoetina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estearoil-CoA Dessaturase/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
Sepsis remains the most important cause of acute kidney injury (AKI) and acute lung injury (ALI) in critically ill patients. The cecal ligation and puncture (CLP) model in experimental mice reproduces most of the clinical features of sepsis. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and pro-angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO protection through the expression of the EPO/EPO receptor (EPO-R) and VEGF/VEF-R2 systems in kidneys and lungs of mice undergoing CLP-induced sepsis. Male inbred Balb/c mice were divided in three experimental groups: Sham, CLP, and CLP+EPO (3000IU/kg sc). Assessment of renal functional parameters, survival, histological examination, immunohistochemistry and/or Western blottings of EPO-R, VEGF and VEGF-R2 were performed at 18h post-surgery. Mice demonstrated AKI by elevation of serum creatinine and renal histologic damage. EPO treatment attenuates renal dysfunction and ameliorates kidney histopathologic changes. Additionally, EPO administration attenuates deleterious septic damage in renal cortex through the overexpression of EPO-R in tubular interstitial cells and the overexpression of the pair VEGF/VEGF-R2. Similarly CLP- induced ALI, as evidenced by parenchymal lung histopathologic alterations, was ameliorated through pulmonary EPO-R, VEGF and VEGF-R2 over expression suggesting and improvement in endothelial survival and functionality. This study demonstrates that EPO exerts protective effects in kidneys and lungs in mice with CLP-induced sepsis through the expression of EPO-R and the regulation of the VEGF/VEGF-R2 pair.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/tratamento farmacológico , Eritropoetina/uso terapêutico , Receptores da Eritropoetina/metabolismo , Sepse/microbiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Lesão Pulmonar Aguda/fisiopatologia , Animais , Nitrogênio da Ureia Sanguínea , Ceco/patologia , Creatinina/sangue , Modelos Animais de Doenças , Eritropoetina/administração & dosagem , Eritropoetina/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Ligadura , Masculino , Camundongos Endogâmicos BALB C , Punções , Análise de SobrevidaRESUMO
Las ciencias básicas, la medicina oral y los nuevos avanzces en biotecnología y bioinformática constituyen un gran campo de investigación dentro de la odontología actual. En este sentido, dichos avances están proporcionandoun nuevo conjunto de estrategias terapéuticas para el manejo clínico de los pacientes con dolencias dentales y craneofaciales. Es importante destacar que las disciplinas relacionadas con las ciencias básicas, la medicina oral, la biotecnología y la bioinformática, han contribuido de manera trascendental al entendimiento de la fisiología y lasdiversas patologías que afectan las condiciones de normalidad del sistema bucal. La ingeniería tisular se considera como un enfoque prometedor para la odontología regenerativa, con el objetivofinal de reemplazar morfológica y funcionalmente los tejidos periodontales y/o los dientes perdidos a través dela síntesis in vitro de sustitutos análogos tisulares, considerando que el diente y las estructuras periodontales son importantes órganos del complejo craneofacial, los tratamientos utilizados para las enfermedades que los afectan no lo restauran completamente. La odontología clínica está incursionando en una nueva era en donde el enfoque terapéutico es el uso de terapia génica, terapia celular, ingeniería tisular y lamedicina regenerativa, ampliando el arsenal de posibilidades para nuestros pacientes. Una línea de investigaciónfundamental en ingeniería tisular y medicina regenerativa son las células madres. Como parte de los nuevos avances de la odontología a nivel mundial, científicos e investigadores del mundo aplican la bioingeniería para lograr reconstrucciones maxilofaciales,regeneraciones óseas y reconstrucciones de piezas dentales a partir de células madre como parte de tratamientos inovadores...
Assuntos
Humanos , Células-Tronco/fisiologia , Odontologia/tendências , Engenharia Tecidual , Técnicas de Cultura de Células/métodos , Bioengenharia/métodos , Células Cultivadas/fisiologia , Células Cultivadas/transplante , Doenças da Boca/terapia , Regeneração Óssea/fisiologia , Tecnologia OdontológicaRESUMO
Paclitaxel, an antitumoral drug, was used in a single dose (29 mg/kg i.p.) as an injury agent for inducing transient suppression of hematopoiesis in a murine experimental model during 10days. The aim of this study focuses on erythropoietin (EPO) receptor, GATA binding protein 1 (globin transcription factor 1) (GATA-1) and erythroid Krüppel-like factor (EKLF) expressions related to the apoptotic events triggered by paclitaxel in bone marrow and the subsequent in vivo erythropoietic recovery. Results showed a massive impairment of erythropoiesis early post paclitaxel administration (1-2 days), which involved induction of high Bax/Bcl-x(L) ratio, caspase-3 activation, disruptions of the medullar niche and cell death by both apoptosis and necrosis. EPO receptor over-expression was noticed from day 3 onwards. It prompted the subsequent up-regulations of GATA-1 and EKLF transcription factors as well as of the anti apoptotic protein Bcl-x(L), crucial proteins in driving erythropoiesis. This study suggests that EPO receptor recovery is necessary for the subsequent bone marrow ability to accomplish the erythroid program through the modulation of apoptotic and survival events after a single paclitaxel insult. These findings contribute to new insights into the molecular mechanisms involved during in vivo erythropoiesis post paclitaxel administration. Therefore, the detailed knowledge of the injury elicited by this drug on red blood cell production may have clinical relevance to explore new therapeutic approaches.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Fator de Transcrição GATA1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Paclitaxel/farmacologia , Receptores da Eritropoetina/metabolismo , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Caspase 3/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hematologia , Radioisótopos de Ferro/metabolismo , Camundongos , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
The present study was conducted to investigate the role of Formocresol (FC)-induced apoptosis and necrotic cell death in murine peritoneal macrophages (pMø). Macrophages were cultured with 1:100 FC for 2 to 24 h. The viability (trypan blue assay), cell morphology (scanning electronic microscope), and apoptotic and necrotic indexes (light and fluorescent microscopy) were determined at different scheduled times. Simultaneously, the expressions of proteins related to stress, survival, and cell death were measured by western blotting. FC-exposed macrophages exhibited maximal apoptosis from 2 to 6 h, coincident with Bax overexpression (P < 0.001). Additionally, Bcl-x(L) showed maximal expression between 12 and 24 h suggesting its survival effect in pMø. The lowest pMø viability and the increment of the necrotic rate from 4 to 12 h were observed in accordance to Fas and Hsp60 overexpressions. In summary, all the experimental data suggest that two different pathways emerge in pMø exposed to FC, one leading Bax-dependent apoptosis (2-6 h) and the other one favoring necrosis (4-18 h), related to Fas-receptor and Hsp60 stress signal.
Assuntos
Apoptose , Formocresóis/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Necrose , Animais , Western Blotting , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 60/biossíntese , Expressão Gênica , Macrófagos Peritoneais/citologia , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Transdução de Sinais/efeitos dos fármacos , Proteína bcl-X/biossínteseRESUMO
Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.
Assuntos
Anemia/sangue , Células da Medula Óssea/citologia , Caspase 3/metabolismo , Eritropoese , Fator de Transcrição GATA1/metabolismo , Receptores da Eritropoetina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Doença Aguda , Anemia/induzido quimicamente , Anemia/metabolismo , Anemia/patologia , Animais , Apoptose , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Precursoras Eritroides/citologia , Feminino , Fluoruracila/farmacologia , Camundongos , Índice MitóticoRESUMO
The effect of a single dose of cyclophosphamide (CY, 150 mg/kg i.p.) on the erythropoiesis using an "in vivo" murine model in a time course protocol (0-10 days) was studied through several experimental approaches. Total and differential bone marrow cellularities, apoptosis (TUNEL assays), bone marrow hematopoietic architecture (scanning electronic microscopy), proliferation (DNA assay), BM erythroid progenitors growth (semisolid clonogenic assays) and protein expressions for erythroid commitment and survival: erythropoietin receptor (EPO-R), Bcl-x(L), Bax (immunoblottings) were performed on the scheduled days. Most of the experiences were conducted comparing spontaneous with human recombinant (hr EPO) "ex vivo" stimulated bone marrow (BM) cells. Erythropoiesis was extremely affected by CY. Maximum apoptosis, minimal cellularities and severe disturbances of BM niche were noticed on the second day. During spontaneous recovery post-CY; EPO-R was expressed between 4 and 5 days. Following BM cells "ex vivo" hr EPO stimulation (2U/ml) EPO-R was expressed throughout the study except the period between the first and fourth day. Bax was noticeable all along the experience with and without hr EPO stimulation. Bcl-x(L) was barely detectable without hr EPO, but its expression showed a gradual enhancement from the fifth day onwards in hr EPO stimulated cells. This fact might be related to the end of the erythroid inhibitory stage and to the recovery of BM EPO-dependent proliferation between the fourth and fifth day, and the further recuperation of BFU-E and CFU-E colonies on days 6 and 7 post-CY, respectively. These findings suggest that the proliferation and differentiation of erythroid progenitor cells after the acute early injury inflicted by CY, is associated with changes in EPO-R expression during spontaneous recovery in this particular experimental system.
Assuntos
Ciclofosfamida/toxicidade , Eritropoese/efeitos dos fármacos , Agonistas Mieloablativos/toxicidade , Receptores da Eritropoetina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/ultraestrutura , Eritropoese/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Intraperitoneais , Masculino , Camundongos , Microscopia Eletrônica de VarreduraRESUMO
Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.