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Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.
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Regulación de la Expresión Génica , Leishmania donovani/genética , Leishmaniasis Visceral/parasitología , Macrófagos/parasitología , Transcriptoma , Animales , Cricetinae , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , Inflamación , Leishmaniasis Visceral/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Mesocricetus , Óxido Nítrico/metabolismo , Fenotipo , Análisis de Secuencia de ARN , Bazo/inmunología , Bazo/parasitología , Regulación hacia ArribaRESUMEN
BACKGROUND This study aims to demonstrate the underlying correlation between the resolution of liver fibrosis induced by Gexia-Zhuyu decoction (GZD) treatment and myeloid cell-mediated angiogenesis. MATERIAL AND METHODS A liver fibrosis mouse model induced by carbon tetrachloride (CCl4) intervention was employed in this study. Dynamics of blood liver function parameters were followed. The liver pathology was detected by Sirius Red and Masson staining. Matrix metalloproteinase (MMP) 2/9, tissue inhibitors of metalloproteinase (TIMP)-1/2, and vascular endothelial growth factor (VEGF)-A expression levels were measured. Bone marrow chimera mice were generated by transfer of bone morrow cells from green fluorescent protein (GFP)-knockin mice into irradiated wild-type mice, and were used it to visualize the role of myeloid cells on the fibrosis resolution induced by GZD treatment. RESULTS The result of Sirius Red and Masson staining and the dynamics of blood liver function parameters showed that 5 weeks of GZD treatment attenuated the severity of liver fibrosis with continual CCl4 administration. GZD treatment promoted the expression of MMP2/9 and repressed the heightened level of TIMP-1/2 in the recovery phase. More notably, the increased VEGF-A and augmented endothelial progenitor cells were observed in the liver and blood in mice that received GZD, and contributed to the remodeling of hepatic vascular though the CXCL12/CXCR4 axis. Then, chimera mice with GFP-positive bone marrow cells were used to show angiogenesis driven by GZD-induced myeloid cell motivation. We found that GZD facilitated myeloid cells binding to the vascular CXCR4 and induced the resolution of fibrosis. CONCLUSIONS This study shows that activation of myeloid cells induced by GZD administration accelerates the functional angiogenesis, which benefits the resolution of CCl4-induced liver fibrosis.
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Medicamentos Herbarios Chinos/farmacología , Cirrosis Hepática/tratamiento farmacológico , Hígado/irrigación sanguínea , Hígado/efectos de los fármacos , Inductores de la Angiogénesis/farmacología , Animales , Células de la Médula Ósea/citología , Tetracloruro de Carbono/farmacología , Modelos Animales de Enfermedad , Femenino , Hígado/química , Hígado/patología , Cirrosis Hepática/inducido químicamente , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/metabolismo , Medicina Tradicional China/métodos , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Neovascularización Patológica/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/análisis , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The activation of hepatic stellate cells (HSCs) is a key event in fibrotic pathogenesis. However, the mechanism involving activation of HSCs in chronic schistosomiasis is not entirely clear. Human HSC LX-2 and human umbilical vein endothelial cells (ECs) were cultured with Schistosoma japonicum antigens (SA) in vitro. Fibrosis-associated genes and cell proliferation were analyzed. HSCs were isolated from mice of chronic schistosomiasis with or without praziquantel (PZQ) treatment, followed by the microarray analysis for the liver fibrosis-associated pathways. Although SA inhibited the activation and proliferation of HSCs, it induced the EC proliferation and vascular endothelial growth factor-a (VEGF) production. VEGF significantly increased the proliferation of HSCs and upregulated the expression of collagen and α-smooth muscle actin. For in vivo study, we found that several fibrosis-associated pathways were involved in the HSCs during the reversal of liver fibrosis caused by schistosomiasis, including VEGF, platelet-derived growth factor, tumor necrosis factor and endothelin-1 pathways. The Ingenuity Pathway Analysis showed that VEGF directly regulated several pro-fibrotic and immune cytokine genes in HSCs, including integrin, fibronectin, interferon-γ, interleukin (IL)-6 and IL-10. Our data indicated the critical role of VEGF signaling in HSC activation in chronic schistosomiasis and highlighted several promising genes and pathways in HSCs as potential targets for therapeutic treatment of liver fibrosis.
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Endotelio Vascular/metabolismo , Células Estrelladas Hepáticas/inmunología , Hígado/patología , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Animales , Antígenos Helmínticos/inmunología , Proliferación Celular , Enfermedad Crónica , Colágeno/metabolismo , Citocinas/metabolismo , Endotelina-1/metabolismo , Endotelio Vascular/inmunología , Femenino , Fibrosis/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hígado/parasitología , Ratones , Ratones Endogámicos BALB C , Esquistosomiasis Japónica/tratamiento farmacológico , TranscriptomaRESUMEN
Cerebral edema is a potentially life-threatening illness, but knowledge of its underlying mechanisms is limited. Here we report that hypobaric hypoxia induces rat cerebral edema and neuronal apoptosis and increases the expression of corticotrophin releasing factor (CRF), CRF receptor type 1 (CRFR1), aquaporin-4 (AQP4), and endothelin-1 (ET-1) in the cortex. These effects, except for the increased expression of CRF itself, could all be blocked by pretreatment with an antagonist of the CRF receptor CRFR1. We also show that, in cultured primary astrocytes: (i) both CRFR1 and AQP4 are expressed; (ii) exogenous CRF, acting through CRFR1, triggers signaling of cAMP/PKA, intracellular Ca(2+), and PKCε; and (iii) the up-regulated cAMP/PKA signaling contributes to the phosphorylation and expression of AQP4 to enhance water influx into astrocytes and produces an up-regulation of ET-1 expression. Finally, using CHO cells transfected with CRFR1(+) and AQP4(+), we show that transfected CRFR1(+) contributes to edema via transfected AQP4(+). In conclusion, hypoxia triggers cortical release of CRF, which acts on CRFR1 to trigger signaling of cAMP/PKA in cortical astrocytes, leading to activation of AQP4 and cerebral edema.
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Acuaporina 4/metabolismo , Edema Encefálico/etiología , Edema Encefálico/metabolismo , Hipoxia/complicaciones , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Apoptosis/genética , Acuaporina 4/genética , Astrocitos/metabolismo , Edema Encefálico/patología , Células CHO , Hormona Liberadora de Corticotropina/metabolismo , Cricetinae , Cricetulus , Endotelina-1/metabolismo , Hipoxia/metabolismo , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Ratas Sprague-Dawley , Transducción de Señal , Transfección , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: High-altitude cerebral edema (HACE) is the severe type of acute mountain sickness (AMS) and life threatening. A subclinical inflammation has been speculated, but the exact mechanisms underlying the HACE are not fully understood. METHODS: Human volunteers ascended to high altitude (3860 m, 2 days), and rats were exposed to hypoxia in a hypobaric chamber (5000 m, 2 days). Human acute mountain sickness was evaluated by the Lake Louise Score (LLS), and plasma corticotrophin-releasing hormone (CRH) and cytokines TNF-α, IL-1ß, and IL-6 were measured in rats and humans. Subsequently, rats were pre-treated with lipopolysaccharide (LPS, intraperitoneal (ip) 4 mg/kg, 11 h) to induce inflammation prior to 1 h hypoxia (7000 m elevation). TNF-α, IL-1ß, IL-6, nitric oxide (NO), CRH, and aquaporin-4 (AQP4) and their gene expression, Evans blue, Na(+)-K(+)-ATPase activity, p65 translocation, and cell swelling were measured in brain by ELISA, Western blotting, Q-PCR, RT-PCR, immunohistochemistry, and transmission electron micrography. MAPKs, NF-κB pathway, and water permeability of primary astrocytes were demonstrated. All measurements were performed with or without LPS challenge. The release of NO, TNF-α, and IL-6 in cultured primary microglia by CRH stimulation with or without PDTC (NF-κB inhibitor) or CP154,526 (CRHR1 antagonist) were measured. RESULTS: Hypobaric hypoxia enhanced plasma TNF-α, IL-1ß, and IL-6 and CRH levels in human and rats, which positively correlated with AMS. A single LPS injection (ip, 4 mg/kg, 12 h) into rats increased TNF-α and IL-1ß levels in the serum and cortex, and AQP4 and AQP4 mRNA expression in cortex and astrocytes, and astrocyte water permeability but did not cause brain edema. However, LPS treatment 11 h prior to 1 h hypoxia (elevation, 7000 m) challenge caused cerebral edema, which was associated with activation of NF-κB and MAPKs, hypoxia-reduced Na(+)-K(+)-ATPase activity and blood-brain barrier (BBB) disruption. Both LPS and CRH stimulated TNF-α, IL-6, and NO release in cultured rat microglia via NF-κB and cAMP/PKA. CONCLUSIONS: Preexisting systemic inflammation plus a short severe hypoxia elicits cerebral edema through upregulated AQP4 and water permeability by TLR4 and CRH/CRHR1 signaling. This study revealed that both infection and hypoxia can cause inflammatory response in the brain. Systemic inflammation can facilitate onset of hypoxic cerebral edema through interaction of astrocyte and microglia by activation of TLR4 and CRH/CRHR1 signaling. Anti-inflammatory agents and CRHR1 antagonist may be useful for prevention and treatment of AMS and HACE.
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Mal de Altura/fisiopatología , Edema Encefálico/etiología , Edema Encefálico/fisiopatología , Hipoxia/complicaciones , Hipoxia/fisiopatología , Inflamación/fisiopatología , Adolescente , Animales , Barrera Hematoencefálica/efectos de los fármacos , Agua Corporal/metabolismo , Permeabilidad de la Membrana Celular , Hormona Liberadora de Corticotropina/sangre , Citocinas/sangre , Voluntarios Sanos , Humanos , Lipopolisacáridos , Masculino , Ratas , Ratas Sprague-Dawley , Adulto JovenRESUMEN
BACKGROUND: The airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-ß (TGFß) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. RESULTS: Generalized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-κB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-κB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFß stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFß-induced the NF-κB/RelA pathway by observing a 2-fold change in NF-κB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFß-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression. CONCLUSIONS: These data indicate that NF-κB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.
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Transición Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , Factor de Transcripción ReIA/genética , Factor de Crecimiento Transformador beta/genética , Células Epiteliales/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/patología , FN-kappa B/genética , Transducción de Señal/genética , Células Madre/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Nanocarrier preparations could effectively improve the utilization rate of pesticides, and reduce pesticide loss. In this study, glyphosate (GLY)-loaded MgAl layered double hydroxide (GLY@LDH) was synthesized via an in-situ method. Subsequently, GLY@LDH composite samples were prepared using a layer-by-layer self-assembly approach and modified with poly-L-aspartic acid (PASP) and chitosan (CS). XRD, FT-IR, SEM, and Zeta potential characterization confirmed that GLY was successfully loaded in the interlayer of LDHs and PASP/CS were successfully encapsulated on the surface of the composite sample. The release effect in different ionic solutions and soils was studied and analyzed. The release behavior conforms to the Ritger-Peppas kinetic model, and the release mechanism was ion exchange, which was further explored by means of XRD, SEM, and molecular simulation. The results of the anti-scouring experiment and contact angle measurement indicated that the layered self-assembly material enhanced the washing resistance of the material. The practical application effect of the sample was verified through a pot experiment. This study provides new insights into the simple preparation of pesticide-controlled release formulations that reduce leaching losses.
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Quitosano , Herbicidas , Plaguicidas , Ácido Aspártico , Preparaciones de Acción Retardada , Espectroscopía Infrarroja por Transformada de Fourier , Hidróxidos , GlifosatoRESUMEN
HIF-1 is composed of HIF-1α and HIF-1ß subunits. It promotes target genes transcription under hypoxia and plays essential roles in cell development, physiological adaptations, and pathological processes. In the past 10 years, the research on signaling pathways of HIF-1 in response to cell hypoxia stress, especially on HIF-1α-mediated gene transcription has made great progress.
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Factor 1 Inducible por Hipoxia/metabolismo , Transducción de Señal , Animales , Hipoxia de la Célula/fisiología , HumanosRESUMEN
BACKGROUND: The disruption of the blood brain barrier (BBB) is the key factor leading to neurological impairment after intracerebral hemorrhage (ICH) injury. Adiponectin receptor 1 (AdipoR1) has an important effect contributing to the integrity of BBB. As a homologue of adiponectin, recombinant C1q/TNF-related protein 9 (rCTRP9) has neuroprotective effect in cerebrovascular diseases. The aim of this study was to investigate the protective effect of AdipoR1 activation with rCTRP9 on BBB integrity after ICH injury and the potential mechanisms. METHODS: 177 male mice were subjected in this study. ICH was induced by injecting collagenase into the right basal ganglia. rCTRP9 was treated intranasally at 1 hour after ICH. Selective siRNA was administered prior to ICH. Western blot, immunofluorescence staining, neurobehavioral tests, and BBB permeability were evaluated. RESULTS: ICH increased the expression of endogenous AdipoR1 and CTRP9. Administration of rCTRP9 ameliorated neurological deficits and reduced the BBB permeability at 24 hours in ICH mice. Furthermore, rCTRP9 promoted the expression of AdipoR1, APPL1, p-AMPK, Nrf2, and tight junctional proteins. The intervention of specific siRNA of AdipoR1, APPL1, and p-AMPK reversed the protective effects of rCTRP9. CONCLUSIONS: Activation of AdipoR1 with rCTRP9 improved neurological functions and preserved BBB integrity through the APPL1/AMPK/Nrf2 signaling pathway in ICH mice. Therefore, CTRP9 could serve as a promising therapeutic method to alleviate BBB injury following ICH in patients.
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Adiponectina/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Hemorragia Cerebral/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Receptores de Adiponectina/agonistas , Proteínas Recombinantes/administración & dosificación , Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Permeabilidad de la Membrana Celular , Hemorragia Cerebral/etiología , Hemorragia Cerebral/patología , Masculino , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de SeñalRESUMEN
Electrospinning is an effective technique to fabricate one-dimensional materials. In this study, cobalt-embedded carbon nanofibers (Co@CNFs) are obtained via carbonization of electrospun cobalt Prussian blue analogue (Co-Co PBA) under nitrogen atmosphere. The Co@CNFs have metallic cobalt surrounded by graphitic carbon shells and possess high specific surface area, rich porosity, high graphitic degree, and rational nitrogen doping. The structure merits endow them with excellent electrocatalytic performances for oxygen reduction reaction (ORR): an onset potential of 0.867 V vs. RHE and 0.784 V vs. RHE at j = - 3 mA cm-2 with a four-electron transfer process. Through a further mild oxidation process, we obtain Co3O4 nanoparticles-embedded nitrogen-doped carbon (Co3O4@CNFs) with spindle-like morphology. When working as the anode materials for lithium-ion batteries (LIBs), Co3O4@CNFs show high specific capacity, good stability, and excellent rate capability. The Co3O4@CNFs anode delivers a discharge specific capacity of 1404 mA h g-1 after 100 cycles at a current density of 100 mA g-1 and about 500 mA h g-1 after 500 cycles at 2000 mA g-1. The diffusion- and capacitive-controlled processes both contribute to the charge storage of the Co3O4@CNFs electrode. This study provides a new strategy to fabricate the excellent electrocatalysts for ORR and anode materials for LIBs via facile electrospinning.
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INTRODUCTION: Deltonin, an active component extracted from Dioscorea zingiberensis C.H. WRIGHT, was widely utilized in traditional Chinese medicines. It has been shown to have anti-cancer functions such as colon cancer, breast cancer, and head and neck squamous carcinoma. Herein, we will investigate the role of deltonin in cerebral ischemia/reperfusion injuries. METHODS: Ly294002 and anisomycin were used as inhibitors to monitor the effects of deltonin. Middle cerebral artery occlusion I/R model was constructed. Infarct volumes, neurological deficits and brain water contents were evaluated under different conditions. Rotarod test, ELISA, and Western blotting were carried to investigate the effects in vitro. RESULTS: We found that deltonin in ischemia/reperfusion (I/R) rats greatly enhanced brain damages as well as neurological functions through up-regulating p-Akt and p-mTOR as well as inhibiting the expressions of LC3-II/LC3-I, Beclin-1, IL-1, TLR4, and p-p38. Deltonin exerted neuroprotection effect through relieving autophagy activity by regulating PI3K/Akt/mTOR signaling. Deltonin suppressed inflammation reactions through modulation TLR4/p38/MAPK signaling as well. CONCLUSION: Overall, our data suggested that deltonin could suppress ischemic brain injury by regulating autophagy and inflammation during I/R. Deltonin can be a potential therapeutic method for patient with I/R.
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Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.
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Linfocitos T CD4-Positivos/inmunología , Leishmaniasis Visceral/inmunología , Macrófagos/inmunología , Animales , Técnicas de Cocultivo , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Inmunofenotipificación , Leishmania donovani/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/parasitología , Activación de Linfocitos/genética , Activación de Macrófagos/genética , Masculino , Mesocricetus , Proteína 2 Ligando de Muerte Celular Programada 1/antagonistas & inhibidores , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/inmunologíaRESUMEN
The homeobox encodes a DNA-binding domain found in transcription factors regulating key developmental processes. The most notable examples of homeobox containing genes are the Hox genes, arranged on chromosomes in the same order as their expression domains along the body axis. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Here we report the discovery of a conserved structural signature of the 180-base pair DNA fragment comprising the homeobox. We demonstrate that the homeobox DNA has a characteristic 3-base-pair periodicity in the hydroxyl radical cleavage pattern. This periodic pattern is significant in most of the 39 mammalian Hox genes and in other homeobox-containing transcription factors. The signature is present in segmented bilaterian animals as evolutionarily distant as humans and flies. It remains conserved despite the fact that it would be disrupted by synonymous mutations, which raises the possibility of evolutionary selective pressure acting on the structure of the coding DNA. The homeobox coding DNA may therefore have a secondary function, possibly as a regulatory element. The existence of such element may have important consequences for understanding how these genes are regulated.
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Secuencia Conservada , Genes Homeobox/genética , Animales , Drosophila , Evolución Molecular , Humanos , Ratones , Motivos de Nucleótidos , Sistemas de Lectura AbiertaRESUMEN
Transcriptomic and proteomic approaches have separately proven effective at identifying novel mechanisms affecting addiction-related behavior; however, it is difficult to prioritize the many promising leads from each approach. A convergent secondary analysis of proteomic and transcriptomic results can glean additional information to help prioritize promising leads. The current study is a secondary analysis of the convergence of recently published separate transcriptomic and proteomic analyses of nucleus accumbens (NAc) tissue from rats subjected to environmental enrichment vs. isolation and cocaine self-administration vs. saline. Multiple bioinformatics approaches (e.g. Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA)) were used to interrogate these rich data sets. Although there was little correspondence between mRNA vs. protein at the individual target level, good correspondence was found at the level of gene/protein sets, particularly for the environmental enrichment manipulation. These data identify gene sets where there is a positive relationship between changes in mRNA and protein (e.g. glycolysis, ATP synthesis, translation elongation factor activity, etc.) and gene sets where there is an inverse relationship (e.g. ribosomes, Rho GTPase signaling, protein ubiquitination, etc.). Overall environmental enrichment produced better correspondence than cocaine self-administration. The individual targets contributing to mRNA and protein effects were largely not overlapping. As a whole, these results confirm that robust transcriptomic and proteomic data sets can provide similar results at the gene/protein set level even when there is little correspondence at the individual target level and little overlap in the targets contributing to the effects.
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Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/terapia , Ambiente , Proteoma , Transcriptoma , Animales , Cocaína/administración & dosificación , Biología Computacional , Modelos Animales de Enfermedad , Inhibidores de Captación de Dopamina/administración & dosificación , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas Sprague-Dawley , Autoadministración , Aislamiento SocialRESUMEN
There exists much variability in susceptibility/resilience to addiction in humans. The environmental enrichment paradigm is a rat model of resilience to addiction-like behavior, and understanding the molecular mechanisms underlying this protective phenotype may lead to novel targets for pharmacotherapeutics to treat cocaine addiction. We investigated the differential regulation of transcript levels using RNA sequencing of the rat nucleus accumbens after environmental enrichment/isolation and cocaine/saline self-administration. Ingenuity Pathways Analysis and Gene Set Enrichment Analysis of 14,309 transcripts demonstrated that many biofunctions and pathways were differentially regulated. New functional pathways were also identified for cocaine modulation (e.g., Rho GTPase signaling) and environmental enrichment (e.g., signaling of EIF2, mTOR, ephrin). However, one novel pathway stood out above the others, the retinoic acid (RA) signaling pathway. The RA signaling pathway was identified as one likely mediator of the protective enrichment addiction phenotype, an interesting result given that nine RA signaling-related genes are expressed selectively and at high levels in the nucleus accumbens shell (NAcSh). Subsequent knockdown of Cyp26b1 (an RA degradation enzyme) in the NAcSh of rats confirmed this role by increasing cocaine self-administration as well as cocaine seeking. These results provide a comprehensive account of enrichment effects on the transcriptome and identify RA signaling as a contributing factor for cocaine addiction.
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We have shown that hypoxia reduces plasma insulin, which correlates with corticotropin-releasing hormone (CRH) receptor 1 (CRHR1) in rats, but the mechanism remains unclear. Here, we report that hypobaric hypoxia at an altitude of 5,000 m for 8 h enhances rat plasma CRH, corticosterone, and glucose levels, whereas the plasma insulin and pancreatic ATP/ADP ratio is reduced. In islets cultured under normoxia, CRH stimulated insulin release in a glucose- and CRH-level-dependent manner by activating CRHR1 and thus the cAMP-dependent protein kinase pathway and calcium influx through L-type channels. In islets cultured under hypoxia, however, the insulinotropic effect of CRH was inactivated due to reduced ATP and cAMP and coincident loss of intracellular calcium oscillations. Serum and glucocorticoid-inducible kinase 1 (SGK1) also played an inhibitory role. In human volunteers rapidly ascended to 3,860 m, plasma CRH and glucose levels increased without a detectable change in plasma insulin. By contrast, volunteers with acute mountain sickness (AMS) exhibited a marked decrease in HOMA insulin sensitivity (HOMA-IS) and enhanced plasma CRH. In conclusion, hypoxia may attenuate the CRH-insulinotropic effect by reducing cellular ATP/ADP ratio, cAMP and calcium influx, and upregulated SGK1. Hypoxia may not affect HOMA-IS in healthy volunteers but reduces it in AMS volunteers.
Asunto(s)
Altitud , Hormona Liberadora de Corticotropina/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Hipoxia/sangre , Hipoxia/metabolismo , Insulina/metabolismo , Adenosina Trifosfato/metabolismo , Adolescente , Adulto , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Calcio/metabolismo , Corticosterona/sangre , Hormona Liberadora de Corticotropina/sangre , AMP Cíclico , Humanos , Hidrocortisona/sangre , Insulina/sangre , Masculino , Páncreas/metabolismo , Pirimidinas/farmacología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Adulto JovenRESUMEN
Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate as well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington's and Alzheimer's diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990.
RESUMEN
High-altitude hypoxia can induce physiological dysfunction and mountain sickness, but the underlying mechanism is not fully understood. Corticotrophin-releasing factor (CRF) and CRF type-i receptors (CRFR1) are members of the CRF family and the essential controllers of the physiological activity of the hypothalamo-pituitary-adrenal (HPA) axis and modulators of endocrine and behavioral activity in response to various stressors. We have previously found that high-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of CRF and CRFR1 in the brain and periphery that include activation of the HPA axis in a time- and dose-dependent manner, impaired or improved learning and memory, and anxiety-like behavioral change. Meanwhile, hypoxia induces dysfunctions of the hypothalamo-pituitary-endocrine and immune systems, including suppression of growth and development, as well as inhibition of reproductive, metabolic and immune functions. In contrast, the small mammals that live on the Qinghai-Tibet Plateau alpine meadow display low responsiveness to extreme high-altitude-hypoxia challenge, suggesting well-acclimatized genes and a physiological strategy that developed during evolution through interactions between the genes and environment. All the findings provide evidence for understanding the neuroendocrine mechanisms of hypoxia-induced physiological dysfunction. This review extends these findings.