RESUMO
Mesenchymal stem cells (MSCs), which are isolated from adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), or bone marrow, have therapeutic potential including anti-inflammatory and immunomodulatory activities. It was recently reported that MSCs are also effective as a therapeutic treatment for neuropathic pain, although the underlying mechanisms have yet to be resolved. Therefore, in this study, we investigated the effects of human AD- and UC-MSCs on neuropathic pain and its mechanisms using rat models of partial sciatic nerve ligation (PSNL). AD- or UC-MSCs were intravenously administered 4 days after PSNL. Antinociceptive effects were then evaluated using the von Frey and weight-bearing tests. We found that, 3-9 days after the administration of AD- or UC-MSCs to PSNL-exposed rats, both the mechanical threshold and differences in weight-bearing of the right and left hind paws were significantly improved. To reveal the potential underlying antinociceptive mechanisms of MSCs, the levels of activation transcription factor 3- and ionized calcium-binding adapter molecule 1-positive cells were measured by immunohistochemical analysis. AD- and UC-MSCs significantly decreased the levels of these proteins that were induced by PSNL in the dorsal root ganglia. Additionally, UC-MSC significantly improved the PSNL-induced decrease in the myelin basic protein level in the sciatic nerve, indicating that UC-MSC reversed demyelination of the sciatic nerve produced by PSNL. These data suggest that AD- and UC-MSCs may help in the recovery of neuropathic pain via the different regulation; AD-MSCs exhibited their effects via suppressed neuronal damage and anti-inflammatory actions, while UC-MSCs exhibited their effects via suppressed neuronal damage, anti-inflammatory actions and remyelination.
Assuntos
Transplante de Células-Tronco Mesenquimais , Neuralgia/terapia , Neurônios/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Tecido Adiposo/citologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/imunologia , Gânglios Espinais/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/cirurgia , Cordão Umbilical/citologiaRESUMO
It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer's patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.
Assuntos
Tecido Adiposo/imunologia , Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunidade nas Mucosas , Células-Tronco Mesenquimais/imunologia , Nódulos Linfáticos Agregados/imunologia , Aloenxertos , Animais , Toxina da Cólera/toxicidade , Feminino , Imunoglobulina A Secretora/imunologia , Interferon gama/imunologia , Interleucina-4/imunologia , Masculino , Transplante de Células-Tronco Mesenquimais , CamundongosRESUMO
Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Absorção Intestinal/fisiologia , Ferro/metabolismo , Manganês/metabolismo , Anemia Hipocrômica/genética , Anemia Hipocrômica/patologia , Animais , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Zinco/metabolismoRESUMO
Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Transportador de Cobre 1 , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ativação Enzimática , Deleção de Genes , MAP Quinase Quinase 1/metabolismo , Camundongos , Fosforilação , Ligação ProteicaRESUMO
Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu(+) importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Estabilidade Proteica , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Cátions/genética , Polaridade Celular , Transportador de Cobre 1 , Dieta , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Ratos , SuínosRESUMO
Copper (Cu) is an essential cofactor for a variety of metabolic functions, and the regulation of systemic Cu metabolism is critical to human health. Dietary Cu is absorbed through the intestine, stored in the liver, and mobilized into the circulation; however, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac-specific knockout of the Ctr1 Cu transporter (Ctr1(hrt/hrt)), resulting in cardiac Cu deficiency and severe cardiomyopathy. Unexpectedly, Ctr1(hrt/hrt) mice exhibited increased serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1(hrt/hrt) mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand, and illuminate a systemic regulation in which the Cu status of the heart is signaled to organs that take up and store Cu.
Assuntos
Cobre/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Adenosina Trifosfatases/metabolismo , Animais , Cardiomiopatia Dilatada/etiologia , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cobre/deficiência , Transportador de Cobre 1 , ATPases Transportadoras de Cobre , Drosophila/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Camundongos KnockoutRESUMO
The trace element copper (Cu) is a cofactor for biochemical functions ranging from energy generation to iron (Fe) acquisition, angiogenesis, and free radical detoxification. While Cu is essential for life, the molecules that mediate dietary Cu uptake have not been identified. Ctr1 is a homotrimeric protein, conserved from yeast to humans, that transports Cu across the plasma membrane with high affinity and specificity. Here we describe the generation of intestinal epithelial cell-specific Ctr1 knockout mice. These mice exhibit striking neonatal defects in Cu accumulation in peripheral tissues, hepatic Fe overload, cardiac hypertrophy, and severe growth and viability defects. Consistent with an intestinal Cu absorption block, the growth and viability defects can be partially rescued by a single postnatal Cu administration, indicative of a critical neonatal metabolic requirement for Cu that is provided by intestinal Ctr1. These studies identify Ctr1 as the major factor driving intestinal Cu absorption in mammals.