RESUMEN
To investigate the intestinal amino acids pathway in depression-like offspring rats induced by maternal separation. Sprague-Dawley (SD) female rats were randomly divided into a control group (=8) and a maternal separation group (=8). After normal delivery, the maternal rats were separated from offsprings for 14 consecutive days and 3â h per day in maternal separation group; while rats in the control group was received no interventions in postpartum. Depression-like behaviors of offspring rats were evaluated using the sucrose preference test, novelty suppressed feeding test, and forced swimming test. Amino acid analyzer was used to detect the changes of amino acid contents in the small intestine, and the expressions of alanine-serine-cysteine transporter 2 (ASCT2), solute carrier superfamily 6 member 19 (BAT1) and L-type amino acid transporter 1(LAT1) were detected by Western blot. The weight of the offspring rats in the maternal separation group was significantly lower than that of the control group at 21 and 28â d (=4.925 and 5.766, all <0.01). Compared with the control group, the percentage of sucrose preference of the offspring rats in the maternal separation group was significantly reduced (=2.709, <0.05), and the feeding latency was significantly prolonged (=-13.431, <0.01). The immobility time in FST of maternal separation group was significantly longer (=-3.616, <0.01).Increased concentration of aspartic acid (=-6.672, <0.01) and down-regulation of glutamine (=3.107, <0.01) and glycine (=9.781, <0.01) were observed in maternal separation group. Western blot analysis revealed that the protein expressions of ASCT2 (=6.734, <0.01) and BAT1 (=9.015, <0.01) in maternal separation group were reduced, while the expression of LAT1 was increased (=-8.942, <0.01). Maternal separation can induce the depression-like behavior in offspring rats; the amino acid contents and the amino acid transporter expression in the small intestine are reduced, which may be related to depression-like behavior induced by maternal separation.
Asunto(s)
Depresión , Privación Materna , Aminoácidos , Animales , Depresión/etiología , Femenino , Hipocampo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navß4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navß4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navß4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navß4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navß4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navß2 nor overexpression of a Navß4-mutant, predicted to be an inactive form of Navß4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navß4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navß4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navß4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navß4 as an important regulator of INaR and excitability in sensory neurons. As such, Navß4 is a potential target for the manipulation of pain sensations.
Asunto(s)
Activación del Canal Iónico , Células Receptoras Sensoriales/metabolismo , Subunidad beta-4 de Canal de Sodio Activado por Voltaje/metabolismo , Secuencia de Aminoácidos , Animales , Ganglios Espinales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Ratas Sprague-Dawley , Subunidad beta-2 de Canal de Sodio Activado por Voltaje , Subunidad beta-4 de Canal de Sodio Activado por Voltaje/químicaRESUMEN
Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple-negative breast cancer (TNBC), estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in approximately 37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Because RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects. SIGNIFICANCE: Mutant RAS- and PIK3CA-driven breast cancers distinctly affect the function of skeletal muscle. Therefore, research and therapeutic targeting of cancer-induced systemic effects need to take aberrant cancer genome into consideration.
Asunto(s)
Músculo Esquelético , Humanos , Femenino , Animales , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ratones , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Mutación , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Peripheral nerve injury causes spontaneous and long-lasting pain, hyperalgesia, and allodynia. Excitatory amino acid receptor-dependent increases in descending facilitatory drive from the brainstem rostral ventromedial medulla (RVM) contribute to injury-evoked hypersensitivity. Although increased excitability likely reflects changes in synaptic efficacy, the cellular mechanisms underlying injury-induced synaptic plasticity are poorly understood. Neuronal pentraxin 1 (NP1), a protein with exclusive CNS expression, is implicated in synaptogenesis and AMPA receptor recruitment to immature synapses. Its role in the adult brain and in descending pain facilitation is unknown. Here, we use the spared nerve injury (SNI) model in rodents to examine this issue. We show that SNI increases RVM NP1 expression and constitutive deletion or silencing NP1 in the RVM, before or after SNI, attenuates allodynia and hyperalgesia in rats. Selective rescue of RVM NP1 expression restores behavioral hypersensitivity of knock-out mice, demonstrating a key role of RVM NP1 in the pathogenesis of neuropathic pain.
Asunto(s)
Proteína C-Reactiva/antagonistas & inhibidores , Proteína C-Reactiva/fisiología , Hiperalgesia/metabolismo , Hiperalgesia/prevención & control , Bulbo Raquídeo/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Neuralgia/metabolismo , Neuralgia/prevención & control , Animales , Proteína C-Reactiva/genética , Silenciador del Gen/fisiología , Hiperalgesia/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuralgia/genética , Neuronas/metabolismo , Manejo del Dolor/métodos , Dimensión del Dolor/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.
RESUMEN
BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca²âº channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca²âº channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Canales de Calcio Tipo N/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Proteínas del Tejido Nervioso/química , Nocicepción , Nociceptores/metabolismo , Péptidos/uso terapéutico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Secuencia de Aminoácidos , Animales , Separación Celular , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Hiperalgesia/complicaciones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neurotransmisores/metabolismo , Nocicepción/efectos de los fármacos , Nociceptores/efectos de los fármacos , Nociceptores/patología , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Enfermedades del Sistema Nervioso Periférico/etiología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.
Asunto(s)
Síndromes de Compresión Nerviosa/prevención & control , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas Aferentes/metabolismo , Recuperación de la Función/fisiología , Raíces Nerviosas Espinales/lesiones , Animales , Modelos Animales de Enfermedad , Esquema de Medicación , Estudios de Seguimiento , Masculino , Síndromes de Compresión Nerviosa/tratamiento farmacológico , Compresión Nerviosa , Factores de Crecimiento Nervioso/administración & dosificación , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/administración & dosificación , Neuronas Aferentes/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Raíces Nerviosas Espinales/efectos de los fármacos , Raíces Nerviosas Espinales/metabolismo , Factores de TiempoRESUMEN
The corrosion of 316SS in contact with lead complicates the realization of high coolant temperature. To explore the corrosion behavior at high temperature, the corrosion test of 316SS was performed in liquid lead at a high temperature of 650 °C under Ar with oxygen levels of 10-2 wt% and 10-5 wt% by the static corrosion method. The mass changes after corrosion were determined; then, the corrosion depth and the oxide product formed were further characterized. A multi-oxide layer was formed on the 316SS alloy surface, and the thickness reached 17.5 µm over a period of 100 h at the oxygen level of â¼10-2 wt%. Fe oxide was the main product in the outer layer; the dense Fe-Cr oxide was formed in the inner layer and lead was isolated from the metal substrate. When the oxygen content was 10-5 wt%, corrosion by dissolution at a rather high rate was dominant, and the corrosion depth was as high as 50 µm for 100 h. It is speculated that the oxide layer is also formed at the initial stage but gets dissociated when there is no oxygen supply to sustain the oxide layer with prolonged exposure time. The oxygen content in the cover gas greatly influences the corrosion behavior of 316SS, thus directly affecting the application of 316SS immersed in liquid lead at high temperature.
RESUMEN
miR-486 is a myogenic microRNA, and its reduced skeletal muscle expression is observed in muscular dystrophy. Transgenic overexpression of miR-486 using muscle creatine kinase promoter (MCK-miR-486) partially rescues muscular dystrophy phenotype. We had previously demonstrated reduced circulating and skeletal muscle miR-486 levels with accompanying skeletal muscle defects in mammary tumor models. To determine whether skeletal muscle miR-486 is functionally similar in dystrophies and cancer, we performed functional limitations and biochemical studies of skeletal muscles of MMTV-Neu mice that mimic HER2+ breast cancer and MMTV-PyMT mice that mimic luminal subtype B breast cancer and these mice crossed to MCK-miR-486 mice. miR-486 significantly prevented tumor-induced reduction in muscle contraction force, grip strength, and rotarod performance in MMTV-Neu mice. In this model, miR-486 reversed cancer-induced skeletal muscle changes, including loss of p53, phospho-AKT, and phospho-laminin alpha 2 (LAMA2) and gain of hnRNPA0 and SRSF10 phosphorylation. LAMA2 is a part of the dystrophin-associated glycoprotein complex, and its loss of function causes congenital muscular dystrophy. Complementing these beneficial effects on muscle, miR-486 indirectly reduced tumor growth and improved survival, which is likely due to systemic effects of miR-486 on production of pro-inflammatory cytokines such as IL-6. Thus, similar to dystrophy, miR-486 has the potential to reverse skeletal muscle defects and cancer burden.
RESUMEN
[This corrects the article DOI: 10.1016/j.omtn.2022.03.009.].
RESUMEN
Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.
RESUMEN
OBJECTIVE: Identification of the neural mechanisms underlying medication overuse headache resulting from triptans. METHODS: Triptans were administered systemically to rats by repeated intermittent injections or by continuous infusion over 6 days. Periorbital and hind paw sensory thresholds were measured to detect cutaneous allodynia. Immunofluorescent histochemistry was employed to detect changes in peptidic neurotransmitter expression in identified dural afferents. Enzyme-linked immunoabsorbent assay was used to measure calcitonin gene-related peptide (CGRP) levels in blood. RESULTS: Sustained or repeated administration of triptans to rats elicited time-dependent and reversible cutaneous tactile allodynia that was maintained throughout and transiently after drug delivery. Triptan administration increased labeling for CGRP in identified trigeminal dural afferents that persisted long after discontinuation of triptan exposure. Two weeks after triptan exposure, when sensory thresholds returned to baseline levels, rats showed enhanced cutaneous allodynia and increased CGRP in the blood following challenge with a nitric oxide donor. Triptan treatment thus induces a state of latent sensitization characterized by persistent pronociceptive neural adaptations in dural afferents and enhanced responses to an established trigger of migraine headache in humans. INTERPRETATION: Triptans represent the treatment of choice for moderate and severe migraine headaches. However, triptan overuse can lead to an increased frequency of migraine headache. Overuse of these medications could induce neural adaptations that result in a state of latent sensitization, which might increase sensitivity to migraine triggers. The latent sensitization could provide a mechanistic basis for the transformation of migraine to medication overuse headache.
Asunto(s)
Cefaleas Secundarias/fisiopatología , Trastornos Migrañosos/tratamiento farmacológico , Triptaminas/toxicidad , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/sangre , Esquema de Medicación , Duramadre/fisiopatología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Masculino , Donantes de Óxido Nítrico/farmacología , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Factores de Tiempo , Nervio Trigémino/efectos de los fármacos , Nervio Trigémino/fisiopatología , Triptaminas/efectos adversosRESUMEN
The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.
Asunto(s)
Proteínas del Tejido Nervioso/farmacología , Dolor/tratamiento farmacológico , Nervios Espinales/lesiones , Animales , Biomarcadores , Péptido Relacionado con Gen de Calcitonina/efectos de los fármacos , Dinorfinas/efectos de los fármacos , Masculino , Ratas , Nervios Espinales/efectos de los fármacosRESUMEN
Migraine is a common neurological disorder often treated with triptans. Triptan overuse can lead to increased frequency of headache in some patients, a phenomenon termed medication overuse headache. Previous preclinical studies have demonstrated that repeated or sustained triptan administration for several days can elicit persistent neural adaptations in trigeminal ganglion cells innervating the dura, prominently characterized by increased labelling of neuronal profiles for calcitonin gene related peptide. Additionally, triptan administration elicited a behavioural syndrome of enhanced sensitivity to surrogate triggers of migraine that was maintained for weeks following discontinuation of drug, a phenomenon termed 'triptan-induced latent sensitization'. Here, we demonstrate that triptan administration elicits a long-lasting increase in identified rat trigeminal dural afferents labelled for neuronal nitric oxide synthase in the trigeminal ganglion. Cutaneous allodynia observed during the period of triptan administration was reversed by NXN-323, a selective inhibitor of neuronal nitric oxide synthase. Additionally, neuronal nitric oxide synthase inhibition prevented environmental stress-induced hypersensitivity in the post-triptan administration period. Co-administration of NXN-323 with sumatriptan over several days prevented the expression of allodynia and enhanced sensitivity to stress observed following latent sensitization, but not the triptan-induced increased labelling of neuronal nitric oxide synthase in dural afferents. Triptan administration thus promotes increased expression of neuronal nitric oxide synthase in dural afferents, which is critical for enhanced sensitivity to environmental stress. These data provide a biological basis for increased frequency of headache following triptans and highlight the potential clinical utility of neuronal nitric oxide synthase inhibition in preventing or treating medication overuse headache.
Asunto(s)
Trastornos Migrañosos/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ganglio del Trigémino/efectos de los fármacos , Triptaminas/farmacología , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Duramadre/efectos de los fármacos , Duramadre/fisiopatología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Masculino , Neuronas Aferentes/enzimología , Neuronas Aferentes/fisiología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/fisiopatología , Sumatriptán/administración & dosificación , Sumatriptán/farmacología , Ganglio del Trigémino/fisiopatología , Triptaminas/administración & dosificaciónRESUMEN
Cancer-induced skeletal muscle defects show sex-specific differences in severity with men performing poorly compared to women. Hormones and sex chromosomal differences are suggested to mediate these differences, but the functional skeletal muscle markers to document these differences are unknown. We show that the myogenic microRNA miR-486 is a marker of sex-specific differences in cancer-induced skeletal muscle defects. Cancer-induced loss of circulating miR-486 was more severe in men with bladder, lung, and pancreatic cancers compared to women with the same cancer types. In a syngeneic model of pancreatic cancer, circulating and skeletal muscle loss of miR-486 was more severe in male mice compared to female mice. Estradiol (E2) and the clinically used selective estrogen receptor modulator toremifene increased miR-486 in undifferentiated and differentiated myoblast cell line C2C12 and E2-inducible expression correlated with direct binding of estrogen receptor alpha (ERα) to the regulatory region of the miR-486 gene. E2 and toremifene reduced the actions of cytokines such as myostatin, transforming growth factor ß, and tumor necrosis factor α, which mediate cancer-induced skeletal muscle wasting. E2- and toremifene-treated C2C12 myoblast/myotube cells contained elevated levels of active protein kinase B (AKT) with a corresponding decrease in the levels of its negative regulator PTEN, which is a target of miR-486. We propose an ERα:E2-miR-486-AKT signaling axis, which reduces the deleterious effects of cancer-induced cytokines/chemokines on skeletal muscle mass and/or function.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/biosíntesis , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Neoplasias/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Estradiol/farmacología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Enfermedades Musculares/complicaciones , Miostatina/biosíntesis , Neoplasias/complicaciones , Factores Sexuales , Transducción de Señal , Toremifeno/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. METHODS: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. RESULTS: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1ß, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. CONCLUSIONS: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.
RESUMEN
NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys²66 and Cys³°9 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.
Asunto(s)
Disulfuros/metabolismo , Proteínas de la Mielina/química , Ingeniería de Proteínas/métodos , Receptores de Superficie Celular/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Humanos , Masculino , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteínas de la Mielina/genética , Receptor Nogo 1 , Estabilidad Proteica , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Raíces Nerviosas Espinales/lesionesRESUMEN
Breast cancer is a disease of a specific organ, but its effects are felt throughout the body. The systemic effects of breast cancer can lead to functional limitations in patients who suffer from muscle weakness, fatigue, pain, fibromyalgia, or many other dysfunctions, which hasten cancer-associated death. Mechanistic studies have identified quite a few molecular defects in skeletal muscles that are associated with functional limitations in breast cancer. These include circulating cytokines such as TNF-α, IL-1, IL-6, and TGF-ß altering the levels or function of myogenic molecules including PAX7, MyoD, and microRNAs through transcriptional regulators such as NF-κB, STAT3, and SMADs. Molecular defects in breast cancer may also include reduced muscle mitochondrial content and increased extracellular matrix deposition leading to energy imbalance and skeletal muscle fibrosis. This review highlights recent evidence that breast cancer-associated molecular defects mechanistically contribute to functional limitations and further provides insights into therapeutic interventions in managing functional limitations, which in turn may help to improve quality of life in breast cancer patients.
RESUMEN
OBJECTIVE: To explore the pathogenic role of changes of Wnt/ß-catenin signaling pathway in the hippocampus in depression- and anxiety-like behaviors caused by prenatal stress (PS) in offspring rats. METHODS: Twelve female SpragueDawley rats weighing 240-260 g were randomly divided into control and restraint stress groups. The rats in the control group received no interventions, and those in restraint stress group were subjected to restraint stress (three times a day, 45 min each time) at the gestational age of 14-20 days. The 1-month-old offspring rats underwent open field test and forced swimming test to assess the anxiety- and depression-like behaviors, and the expressions of Wnt1, Gsk-3ß and ß-catenin in the hippocampus were detected using Western blotting. RESULTS: In open field test, the offspring rats with PS showed significantly decreased crossings of the center (P < 0.01) with reduced time spent in the center (P < 0.05) compared with control offspring rats. In forced swimming test, the offspring rats in PS group exhibited a significantly longer immobility time than in the control rats, and showed obvious depression- and anxiety-like behaviors. Compared with those in the control offspring rats, Gsk-3ß expression increased significantly while the expressions of ß-catenin and Wnt1 were significantly lowered in the hippocampus of the offspring rats in PS group (P < 0.01). CONCLUSIONS: PS causes changes in Wnt/ß-catenin signaling pathway in the hippocampus to contribute to the occurrence of depression-and anxiety-like behaviors in rats.
Asunto(s)
Ansiedad/metabolismo , Depresión/metabolismo , Hipocampo/metabolismo , Estrés Psicológico/complicaciones , Vía de Señalización Wnt , Animales , Ansiedad/etiología , Conducta Animal , Depresión/etiología , Femenino , Glucógeno Sintasa Quinasa 3 beta , Embarazo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Restricción Física/psicología , Natación/psicologíaRESUMEN
It has been uncovered that chemical dyes are illegally used in traditional Chinese medicines to brighten color and cover up inferiority, which threaten the safety of patients. In the present study, an HPTLC-MS method was developed for the effective screening of 11 chemical dyes (Sudan I, II, III, and IV; 808 Scarlet; Sudan Red 7B; malachite green; Basic Orange 2; auramine; Orange II; and erythrosine) in traditional Chinese medicine (TCM) raw materials and Chinese patent medicines. Firstly, unwashed HPTLC plates were chosen by comparing the background signals of the TLC plates used directly and prewashed with analytical grade and HPLC grade solvents. Twice developments were conducted to isolate chemical dyes of different polarity. Possible adulterants were preliminarily identified by comparing Rf values and in situ UV-Vis spectra with those of the references. Further confirmation was conducted by tandem MS analysis via an elution head-based TLC-MS interface. Sudan I and IV, 808 Scarlet, and Orange II were successfully detected in eight batches of TCM. The proposed method could be applied as a reliable technology for the screening of chemical dyes in TCM.