RESUMO
BACKGROUND: Migraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15% of the world's population being 2-3 times more prevalent in females. The calcitonin gene-related peptide (CGRP) is a key mediator in the pathophysiology of migraine, and a significant advance in the field has been the development of anti-CGRP therapies. The trigeminal ganglion (TG) is thought to be an important site of action for these drugs. Moreover, experimental migraine can be induced by CGRP injection in the TG. The signaling pathway induced by CGRP in the TG is not fully understood, but studies suggest that voltage-gated calcium channels contribute to CGRP effects relevant to migraine. OBJECTIVE: We hypothesised that CGRP injection in the TG enhances CaV3.2 T-type calcium channel currents to contribute to the development of periorbital mechanical allodynia. RESULTS: A Co-Immunoprecipitation assay in tsA-201 cells revealed that CaV3.2 channels form a complex with RAMP-1, a component of the CGRP receptor. Constitutive CGRPR activity was able to inhibit CaV3.2 channels and induce a depolarizing shift in both activation and inactivation curves. Incubation of TG neurons with CGRP increased T-type current density by ~ 3.6 fold, an effect that was not observed in TG neurons from CaV3.2 knockout mice. Incubation of TG neurons with Z944, a pan T-type channel blocker, resulted in an approximately 80% inhibition of T-type currents. In vivo, this treatment abolished the development of periorbital mechanical allodynia induced by CGRP in male and female mice. Likewise, CaV3.2 knockout mice did not develop periorbital mechanical allodynia after intraganglionic CGRP injection. Finally, we demonstrated that the CGRP effect depends on the activation of its canonical GPCR, followed by protein kinase A activation. CONCLUSION: The present study suggests that CGRP modulates CaV3.2 in the TG, an effect possibly mediated by the canonical CGRP receptor and PKA activation. The increase in T-type currents in the TG may represent a contributing factor for the initiation and maintenance of the headache pain during migraine.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Canais de Cálcio Tipo T , Modelos Animais de Doenças , Hiperalgesia , Transtornos de Enxaqueca , Proteína 1 Modificadora da Atividade de Receptores , Gânglio Trigeminal , Animais , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/genética , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/induzido quimicamente , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Hiperalgesia/metabolismo , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Masculino , Camundongos , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Feminino , RatosRESUMO
Inoculation of Bothrops jararaca snake venom (BjV) induces thrombocytopenia in humans and various animal species. Although several BjV toxins acting on hemostasis have been well characterized in vitro, it is not known which one is responsible for inducing thrombocytopenia in vivo. In previous studies, we showed that BjV incubated with metalloproteinase or serine proteinase inhibitors and/or anti-botrocetin antibodies still induced thrombocytopenia in rats and mice. Thus, herein we identified and characterized BjV toxins responsible for inducing thrombocytopenia. Initially, by filtering BjV on ultrafiltration systems, proteins with molecular masses between 30 and 50 kDa were shown to induce thrombocytopenia in mice, but they were not associated with hemorrhagic or coagulating activities. The 50 kDa ultrafiltrate was chromatographed, and two proteins (named fraction D and fraction E) induced thrombocytopenia in mice. However, neither fraction D nor fraction E induced platelet aggregation in platelet-rich plasma or whole blood from humans or mice. By mass spectrometry analysis, fraction E was identified as jararaca glycoprotein Ib (GPIb)-binding protein. Injection of these fractions caused thrombocytopenia in control or Vwf-/- mice, showing that the axis platelet GPIb - von Willebrand factor is not involved in their biological action in vivo. New studies are necessary to understand how these proteins act in vivo.
Assuntos
Bothrops , Trombocitopenia , Animais , Trombocitopenia/induzido quimicamente , Trombocitopenia/metabolismo , Camundongos , Humanos , Bothrops/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Masculino , Veneno de Bothrops jararaca/metabolismo , Mordeduras de Serpentes/metabolismo , Fator de von Willebrand/metabolismo , Venenos de Crotalídeos/toxicidade , Camundongos Knockout , Feminino , Bothrops jararacaRESUMO
The Complement System is composed of more than 40 proteins that act in innate and adaptive immunity. C3 is the most abundant one and C3-deficient patients are more susceptible to recurrent and severe infections. Several studies have demonstrated the importance of C3 in controlling infections. However, its role in leukocyte biology is still poorly understood. This study aimed to evaluate several cellular parameters in macrophages from C3-deficient mice and compare them to similar cells from wild-type counterparts. We observed that in the absence of C3, the population of F4/80low macrophages in the peritoneal cavity of thioglycolate-treated mice is diminished, probably due to the lack of chemotactic factors like C3a and low levels of C5a. Using fluorescence microscopy analysis, we observed that macrophages from C3-deficient mice exhibited morphological alterations when compared to similar cells from wild-type mice. We observed a significant increase in the expression of CD11c, which is part of CR4 (CD11c/CD18), in macrophages from C3-deficient compared to cells from wild-type mice. Treatment with 12-o-tetradecanoylphorbol-13-acetate, stimulated ROS production and MAPK activation by macrophages. However, these parameters were lower in macrophages from C3-deficient mice when compared to wild-type counterparts. In addition, the phagocytosis of iC3b-opsonized Zymosan particles was diminished in macrophages from C3-deficient mice. Our results suggest that C3 deficiency in C57Black/6 mice may influence specific morphological and functional parameters of macrophages, cells of fundamental importance for both the innate and acquired immune responses.
Assuntos
Complemento C3 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio , Animais , Camundongos , Complemento C3/imunologia , Complemento C3/deficiência , Complemento C3/genética , Complemento C3/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Antígeno CD11c/metabolismo , Fenótipo , Fagocitose , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Complemento C5a/imunologia , Complemento C5a/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Antígenos CD11RESUMO
BACKGROUD: abnormalities or defects in oocyte meiosis can result in decreased oocyte quality, reduced ovarian reserve, and female diseases. However, the mechanisms of oocyte meiosis remain largely unknown, especially epigenetic regulation. Here, we explored the role of EZH1/2 (histone methyltransferase of H3K27) in mouse oocyte meiosis by inhibiting its activity and deleting its gene. RESULTS: with embryonic ovary cultured in vitro, EZH1/2 was demonstrated to be essential for oocyte development during meiosis prophase I in mice. Activity inhibition or gene knockout of EZH1/2 resulted in cell apoptosis and a reduction in oocyte numbers within embryonic ovaries. By observing the expression of some meiotic marker protein (γ-H2AX, diplotene stage marker MSY2 and synapsis complex protein SCP1), we found that function deficiency of EZH1/2 resulted in failure of DNA double-strand breaks (DSBs) repair and break of meiotic progression in fetal mouse ovaries. Moreover, Ezh1/2 deficiency led to the suppression of ATM (Ataxia Telangiectasia Mutated kinase) phosphorylation and a decrease in the expression of key DNA repair proteins Hormad1, Mre11, Rad50, and Nbs1 in fetal mouse ovaries, underscoring the enzyme's pivotal role in initiating DNA repair. RNA-seq analysis revealed that Ezh1/2-deletion induced abnormal expression of multiple genes involved into several function of oocyte development in embryonic ovaries. Knockout of Ezh1/2 in ovaries also affected the levels of H3K9me3 and H4K20me2, as well as the expression of their target genes L3mbtl4 and Fbxo44. CONCLUSIONS: our study demonstrated that EZH1/2 plays a role in the DSBs repair in oocyte meiosis prophase I via multiple mechanisms and offers new insights into the physiological regulatory role of histone modification in fetal oocyte guardianship and female fertility.
Assuntos
Prófase Meiótica I , Oócitos , Animais , Feminino , Prófase Meiótica I/fisiologia , Camundongos , Complexo Repressor Polycomb 2/fisiologia , Complexo Repressor Polycomb 2/genética , Camundongos KnockoutRESUMO
BACKGROUND: Excitotoxicity is a process in which NADPH oxidase-2 (NOX-2) plays a pivotal role in the generation of reactive oxygen species (ROS). Oxidative stress influences the expression of Aquaporin 4 (AQP4), a water channel implicated in blood-brain barrier (BBB) permeability and edema formation. The endocannabinoid system is widely distributed in the brain, particularly through the cannabinoid receptor type 1 (CB1) and type 2 (CB2), which have been shown to have a neuroprotective function in brain injury. Given the significant involvement of NOX-2 in ROS production during excitotoxicity, our research aims to assess the participation of NOX-2 in the neuroprotective effect of the cannabinoid receptor agonist WIN55,212-2 against glutamate-induced excitotoxicity damage in the striatum using in vivo model. METHODS: Wild-type mice (C57BL/6) and NOX-2 KO (gp91Cybbtm1Din/J) were stereotactically injected in the striatum with monosodium glutamate or vehicle. Subsequently, a group of mice was administered an intraperitoneal dose of WIN55,212-2, AM251, or AM251/WIN55,212-2 following the intracerebral injection. Motor activity was assessed, and the lesion was examined through histological sections stained with cresyl violet. Additionally, brain water content and Evans blue assay were conducted. The activity of NOX was quantified, and the protein expression of CB1, gp91phox, AQP4, Iba-1, TNF-α, and NF-κB was analyzed using Western blot. Furthermore, ROS formation was measured through the DHE assay. RESULTS: The activation of the endocannabinoid receptors demonstrated a neuroprotective response during excitotoxicity, meditated by NOX-2. The reduction in ROS production led to a decrease in neuroinflammation, and AQP4 expression, resulting in reduced edema formation, and BBB permeability. CONCLUSIONS: During excitotoxic damage, WIN55,212-2 inhibits NOX-2-induced ROS production, reducing brain injury.
Assuntos
Benzoxazinas , Ácido Glutâmico , Camundongos Endogâmicos C57BL , Morfolinas , NADPH Oxidase 2 , Naftalenos , Estresse Oxidativo , Receptor CB1 de Canabinoide , Animais , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , NADPH Oxidase 2/metabolismo , Benzoxazinas/farmacologia , Morfolinas/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Naftalenos/farmacologia , Camundongos , Masculino , Camundongos Knockout , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Espécies Reativas de Oxigênio/metabolismo , Aquaporina 4/metabolismo , Edema Encefálico/metabolismo , Edema Encefálico/prevenção & controleRESUMO
Mesoamerican nephropathy (MeN) is a progressive kidney disease found on the Pacific coast of Central America primarily in young male agricultural workers without typical kidney disease risk factors. While it is generally accepted that environmental exposures contribute to MeN, we hypothesized that there was also an important genetic component. We performed a genome-wide association study comparing individuals with MeN versus individuals with normal kidney function. We found that Native American ancestry was strongly associated with increased risk of MeN. We also identified candidate variants in the OPCML gene, which encodes a protein that binds opioid receptors, that were associated with ~sixfold reduced odds of MeN (allele frequency 0.029 in controls and 0.005 in cases, OR = 0.16; P = 4 × 10-8). Sugarcane workers with the protective OPCML variants had markedly increased urine osmolality suggesting greater ability to defend against hypovolemia. Experiments with Opcml knock-out mice revealed roles for OPCML in fluid balance and temperature regulation consistent with our findings in humans. Our data suggest that heritable differential sensitivity to heat stress and dehydration contributes to high rates of kidney disease in Central America.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Animais , Fatores de Risco , Camundongos , América Central , Camundongos Knockout , Adulto , Nefropatias/genética , Nefropatias/epidemiologia , Feminino , Polimorfismo de Nucleotídeo Único , Doenças Renais Crônicas IdiopáticasRESUMO
Chikungunya virus (CHIKV) infection is characterized by febrile illness, severe joint pain, myalgia, and cardiovascular complications. Given that CHIKV stimulates reactive oxygen species (ROS) and pro- and anti-inflammatory cytokines, events that disrupt vascular homeostasis, we hypothesized that CHIKV induces arterial dysfunction by directly impacting redox-related mechanisms in vascular cells. Wild-type (WT) and iNOS knockout (iNOS-/-) mice were administered either CHIKV (1.0 × 106 PFU/µL) or Mock vehicle via the intracaudal route. In vivo, CHIKV infection induced vascular dysfunction (assessed by a wire myograph), decreased systolic blood pressure (tail-cuff plethysmography), increased IL-6 and IFN-γ, but not TNF-α levels (determined by ELISA), and increased protein content by Western blot. Marked contractile hyporesponsiveness to phenylephrine was observed 48 h post-infection, which was restored by endothelium removal. L-NAME, 1400W, Tiron, and iNOS gene deletion prevented phenylephrine hyporesponsiveness. CHIKV infection increased vascular nitrite concentration (Griess reaction) and superoxide anion (O2â¢-) generation (lucigenin chemiluminescence), and decreased hydrogen peroxide (H2O2, by Amplex Red) levels 48 h post-infection, alongside increased TBARS levels. In vitro, CHIKV infected endothelial cells (EA.hy926) and upregulated ICAM-1 and iNOS protein expression (determined by Western blot). These data support the conclusion that CHIKV-induced alterations in vascular ROS/NF-kB/iNOS/NO signaling potentially contribute to cardiovascular events associated with Chikungunya infection.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Células Endoteliais , Oxirredução , Transdução de Sinais , Animais , Febre de Chikungunya/complicações , Febre de Chikungunya/virologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Camundongos , Vírus Chikungunya/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Masculino , Humanos , Citocinas/metabolismoRESUMO
Patients with glutaric acidemia type I (GA I) manifest motor and intellectual disabilities whose pathogenesis has been so far poorly explored. Therefore, we evaluated neuromotor and cognitive abilities, as well as histopathological and immunohistochemical features in the cerebral cortex and striatum of glutaryl-CoA dehydrogenase (GCDH) deficient knockout mice (Gcdh-/-), a well-recognized model of GA I. The effects of a single intracerebroventricular glutaric acid (GA) injection in one-day-old pups on the same neurobehavioral and histopathological/immunohistochemical endpoints were also investigated. Seven-day-old Gcdh-/- mice presented altered gait, whereas those receiving a GA neonatal administration manifested other sensorimotor deficits, including an abnormal response to negative geotaxis, cliff aversion and righting reflex, and muscle tone impairment. Compared to the WT mice, adult Gcdh-/- mice exhibited motor impairment, evidenced by poor performance in the Rota-rod test. Furthermore, neonatal GA administration provoked long-standing short- and long-term memory impairment in adult Gcdh-/- mice. Regarding the histopathological features, a significant increase in vacuoles and neurodegenerative cells was observed in both the cerebral cortex and striatum of 15- and 60-day-old Gcdh-/- mice and was more pronounced in mice injected with GA. Neuronal loss (decrease of NeuN staining) was also significantly increased in the cerebral cortex and striatum of Gcdh-/- mice, particularly in those neonatally injected with GA. In contrast, immunohistochemistry of MBP, astrocytic proteins GFAP and S100B, and the microglial marker Iba1 was not changed in 60-day-old Gcdh-/- mice, suggesting no myelination disturbance, reactive astrogliosis, and microglia activation, respectively. These data highlight the neurotoxicity of GA and the importance of early treatment aiming to decrease GA accumulation at early stages of development to prevent brain damage and learning/memory disabilities in GA I patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Córtex Cerebral , Corpo Estriado , Glutaril-CoA Desidrogenase , Camundongos Knockout , Animais , Glutaril-CoA Desidrogenase/deficiência , Glutaril-CoA Desidrogenase/genética , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Corpo Estriado/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Camundongos , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Cognição/fisiologia , Cognição/efeitos dos fármacos , Encefalopatias Metabólicas/patologia , Encefalopatias Metabólicas/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
Using genetically engineered mice and cell lines derived from genetically engineered mice we show that depletion of ER delimited Ca2+ stores activates heteromeric Ca2+ entry (SOCE) channels formed obligatorily, but not exclusively by Orai1 molecules. Comparison of Orai-dependent Ca2+ entries revealed Orai1 to be dominant when compared to Orai2 and Orai3. Unexpectedly, we found that store-depletion-activated Ca2+ entry does not depend obligatorily on functionally intact TRPC molecules, as SOCE monitored with the Fura2 Ca2+ reporter dye is unaffected in cells in which all seven TRPC coding genes have been structurally and functionally inactivated. Unexpectedly as well, we found that TRPC-independent Gq-coupled receptor-operated Ca2+ entry (ROCE) also depends on Orai1. Biophysical measurements of Ca2+ release activated Ca2+ currents (Icrac) are likewise unaffected by ablation of all seven TRPC genes. We refer to mice and cells carrying the seven-fold disruption of TRPC genes as TRPC heptaKO mice and cells. TRPC heptaKO mice are fertile allowing the creation of a new homozygous inbred strain.
Assuntos
Cálcio , Canais de Cátion TRPC , Animais , Camundongos , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/genética , Cálcio/metabolismo , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Sinalização do Cálcio , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Camundongos KnockoutRESUMO
Dengue disease is a major problem worldwide, impacting millions of people annually with no specific approved treatments. The pathogenesis of dengue is a complex interplay of viral and host factors, driven in particular by an excessive inflammatory response triggered by the infection. While it has been observed that various viruses can modulate the PI3K/Akt signaling pathway to aid replication and theunderlying mechanisms remainunclear. The study aims to explore the impact of PI3Kγ inhibition during Dengue virus (DENV) infection in vivo. Experiments were performed using both wild-type (WT) and PI3Kγ knockout mice inoculated with DENV. Parameters, including survival rates, hematologic, virologic, histopathologic, and inflammatory analyzes, were evaluated. Additionally, the therapeutic potential of a selective PI3Kγ inhibitor (AS605240) was investigated in DENV-infected A129 mice. PI3Kγ deficiency resulted in lower lethality and provided protection against DENV-induced thrombocytopenia, decreased hemoconcentration, vascular permeability, and liver damage compared to DENV-infected WT littermates. In addition, PI3Kγ deficiency correlated with reduced viral replication in the blood, spleen and liver alongside decreased production of inflammatory mediators in plasma and spleen. Pharmacologic inhibition of PI3Kγ not only ameliorated DENV-induced thrombocytopenia and liver injury, but also reduced DENV replication in target organs. Treatment with AS605240 reduced the concentration of IL-6 in the spleen and plasma.This study sheds light on the significant pro-viral effects of the PI3Kγ signaling pathway during DENV infection and its central role in pathogenesis by curbing excessive DENV-induced inflammation. Inhibition of PI3Kγ shows promising host-directed target for developing novel Dengue disease therapies, offering substantial benefits to hosts.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase , Vírus da Dengue , Dengue , Modelos Animais de Doenças , Camundongos Knockout , Transdução de Sinais , Replicação Viral , Animais , Vírus da Dengue/efeitos dos fármacos , Camundongos , Dengue/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Transdução de Sinais/efeitos dos fármacos , Trombocitopenia/virologia , Fígado/virologia , Fígado/patologia , Interleucina-6/metabolismo , Baço/virologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Quinoxalinas , TiazolidinedionasRESUMO
The 5-lipoxygenase/leukotriene system has been implicated in both physiological and pathological states within the central nervous system. Understanding how this system interacts with the dopaminergic system could provide valuable insights into dopamine-related pathologies. This study focused on examining both motor and non-motor dopamine-related responses in 5-lipoxygenase/leukotriene-deficient mice. We used pharmacological agents such as amphetamine, apomorphine, and reserpine to challenge the dopaminergic system, evaluating their effects on prepulse inhibition reaction (PPI), general motor activity, and oral involuntary movements. Additionally, we analyzed striatal glial marker expression (GFAP and Iba-1) in reserpine-treated mice. The 5-lipoxygenase/leukotriene-deficient mice exhibited increased spontaneous locomotor activity, including both horizontal and vertical exploration, along with stereotyped behavior compared to wild-type mice. This hyperactivity was reduced by acute apomorphine treatment. Although basal PPI responses were unchanged, 5-lipoxygenase/leukotriene-deficient mice displayed a significant reduction in susceptibility to amphetamine-induced PPI disruption. Conversely, these mice were more vulnerable to reserpine-induced involuntary movements. There were no significant differences in the basal expression of striatal GFAP and Iba-1 positive cells between 5-lipoxygenase/leukotriene-deficient and wild-type mice. However, reserpine treatment significantly increased GFAP immunoreactivity in wild-type mice, an effect not observed in 5-lipoxygenase-deficient mice. Additionally, the percentage of activated microglia was significantly higher in reserpine-treated wild-type mice, an effect absents in 5-lipoxygenase/leukotriene-deficient mice. Our findings suggest that 5-lipoxygenase/leukotriene deficiency leads to a distinctive dopaminergic phenotype, indicating that leukotrienes may influence the modulation of dopamine-mediated responses.
Assuntos
Anfetamina , Araquidonato 5-Lipoxigenase , Dopamina , Animais , Masculino , Camundongos , Anfetamina/farmacologia , Apomorfina/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/deficiência , Araquidonato 5-Lipoxigenase/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/deficiência , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Inibição Pré-Pulso/efeitos dos fármacos , Inibição Pré-Pulso/fisiologia , Reserpina/farmacologia , Comportamento Estereotipado/efeitos dos fármacosRESUMO
High neonatal growth hormone (GH) secretion has been described in several species. However, the neuroendocrine mechanisms behind this surge remain unknown. Thus, the pattern of postnatal GH secretion was investigated in mice and rats. Blood GH levels were very high on postnatal day (P)1 and progressively decreased until near zero by P17 in C57BL/6 mice without sex differences. This pattern was similar to that observed in rats, except that female rats showed higher GH levels on P1 than males. In comparison, follicle-stimulating hormone exhibited higher secretion in females during the first 3 weeks of life. Hypothalamic Sst mRNA and somatostatin neuroendocrine terminals in the median eminence were higher in P20/P21 mice than in newborns. Knockout mice for GH-releasing hormone (GHRH) receptor showed no GH surge, whereas knockdown mice for the Sst gene displayed increased neonatal GH peak. Leptin deficiency caused only minor effects on early-life GH secretion. GH receptor ablation in neurons or the entire body did not affect neonatal GH secretion, but the subsequent reduction in blood GH levels was attenuated or prevented by these genetic manipulations, respectively. This phenotype was also observed in knockout mice for the insulin-like growth factor-1 (IGF-1) receptor in GHRH neurons. Moreover, glucose-induced hyperglycemia overstimulated GH secretion in neonatal mice. In conclusion, GH surge in the first days of life is not regulated by negative feedback loops. However, neonatal GH secretion requires GHRH receptor, and is modulated by somatostatin and blood glucose levels, suggesting that this surge is controlled by hypothalamic-pituitary communication.
Assuntos
Animais Recém-Nascidos , Hormônio do Crescimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Somatostatina , Animais , Feminino , Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/sangue , Masculino , Somatostatina/metabolismo , Somatostatina/genética , Camundongos , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Ratos , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Leptina/sangue , Leptina/metabolismo , Hipotálamo/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Hormônio Foliculoestimulante/sangue , Hormônio Foliculoestimulante/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genéticaRESUMO
Growth hormone (GH) action in the brain regulates neuroendocrine axes, energy and glucose homeostasis, and several neurological functions. The lateral hypothalamic area (LHA) contains numerous neurons that respond to a systemic GH injection by expressing the phosphorylated STAT5, a GH receptor (GHR) signaling marker. However, the potential role of GHR signaling in the LHA is unknown. In this study, we demonstrated that â¼70% of orexin- and leptin receptor (LepR)-expressing neurons in the LHA are responsive to GH. Male mice carrying inactivation of the Ghr gene in the LHA were generated via bilateral injections of an adeno-associated virus. In ad libitum-fed mice, GHR ablation in LHA neurons did not significantly change energy and glucose homeostasis. Subsequently, mice were subjected to 5â d of 40% food restriction. Food restriction decreased body weight, energy expenditure, and carbohydrate oxidation. These effects were similarly observed in control and LHAΔGHR mice. While food-deprived control mice progressively increased ambulatory/exploratory activity and food-seeking behavior, LHAΔGHR mice did not show hyperactivity induced by food restriction. GHR ablation in the LHA reduced the percentage of orexin neurons expressing c-Fos during food restriction. Additionally, an acute GH injection increased the expression of c-Fos in LHAORX neurons. Inactivation of Ghr in LepR-expressing cells did not prevent hyperactivity in food-deprived mice, whereas whole-brain Ghr knock-out mice showed reduced ambulatory activity during food restriction. Our findings indicate that GHR signaling in the LHA regulates the activity of orexin neurons and is necessary to increase food-seeking behavior in food-deprived male mice.
Assuntos
Região Hipotalâmica Lateral , Neurônios , Receptores da Somatotropina , Animais , Masculino , Camundongos , Receptores da Somatotropina/metabolismo , Receptores da Somatotropina/genética , Neurônios/metabolismo , Neurônios/fisiologia , Região Hipotalâmica Lateral/metabolismo , Região Hipotalâmica Lateral/fisiologia , Orexinas/metabolismo , Receptores para Leptina/metabolismo , Receptores para Leptina/genética , Camundongos Endogâmicos C57BL , Metabolismo Energético/fisiologia , Comportamento Alimentar/fisiologia , Camundongos Knockout , Privação de Alimentos/fisiologiaRESUMO
Sperm capacitation is a complex process that takes place in the female reproductive tract and empowers mammalian sperm with the competence to fertilize an egg. It consists of an intricate cascade of events that can be mimicked in vitro through incubation in a medium containing essential components, such as bicarbonate, albumin, Ca2+, and energy substrates, among others. Genetic and pharmacological studies have underscored the unique significance of the K+ channel SLO3 in membrane potential hyperpolarization, as evidenced by the infertility of mice lacking its expression. Notably, two key molecular events, sperm hyperpolarization and intracellular alkalinization, are central to the capacitation process. SLO3 is activated by alkalinization. However, the molecular mechanisms responsible for intracellular alkalization and activation of SLO3 are not completely understood. In this study, we examined the impact of Na+/H+ exchangers (NHEs) on mouse sperm membrane hyperpolarization during capacitation. Pharmacological inhibition of the NHE1 blocked membrane hyperpolarization. A similar effect was observed in sperm deficient of the Ca2+ channel CatSper because of NHE1 not being activated by Ca2+. In addition, the sperm-specific NHE (sNHE) KO did not show membrane hyperpolarization upon capacitation or induction with cAMP analogs. Our results show that sNHE is dually modulated by cAMP and membrane hyperpolarization probably through its cyclic nucleotide-binding domain and the voltage-sensor motif, respectively. Together, sNHE and NHE1 provide the alkalinization need for SLO3 activation during capacitation.
Assuntos
Membrana Celular , Potenciais da Membrana , Trocador 1 de Sódio-Hidrogênio , Capacitação Espermática , Espermatozoides , Animais , Masculino , Espermatozoides/metabolismo , Camundongos , Capacitação Espermática/efeitos dos fármacos , Membrana Celular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Camundongos Knockout , Canais de Potássio Ativados por Cálcio de Condutância AltaRESUMO
Colorectal cancer CRC remains one of the leading causes of cancer-related deaths worldwide, with chronic intestinal inflammation identified as a major risk factor. Notably, the tumor suppressor TP53 undergoes mutation at higher rates and earlier stages during human inflammation-driven colon tumorigenesis than in sporadic cases. We investigated whether deleting Trp53 affects inflammation-induced tumor growth and the expression of Lgr5+ cancer stem cells in mice. We examined azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon tumorigenesis in wild-type Trp53 (+/+), heterozygous (+/-), and knockout (-/-) mice. Trp53-/- mice showed increased sensitivity to DSS colitis and earlier accelerated tumorigenesis with 100% incidence. All groups could develop invasive tumors, but knockouts displayed the most aggressive features. Unlike wild-type CRC, knockouts selectively showed increased populations of Lgr5+ colon cancer stem-like cells. Trp53 loss also boosted laminin, possibly facilitating the disruption of the tumor border. This study highlights how Trp53 deletion promotes the perfect storm of inflammation and stemness, driving colon cancer progression. Trp53 deletion dramatically shortened AOM/DSS latency and improved tumor induction efficiency, offering an excellent inflammation-driven CRC model.
Assuntos
Azoximetano , Carcinogênese , Colite , Neoplasias Colorretais , Sulfato de Dextrana , Camundongos Knockout , Células-Tronco Neoplásicas , Receptores Acoplados a Proteínas G , Proteína Supressora de Tumor p53 , Animais , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Camundongos , Azoximetano/toxicidade , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colite/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/etiologia , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Deleção de GenesRESUMO
To further understand the impact of deficiency of the autoimmune regulator (Aire) gene during the adhesion of medullary thymic epithelial cells (mTECs) to thymocytes, we sequenced single-cell libraries (scRNA-seq) obtained from Aire wild-type (WT) (Airewt/wt ) or Aire-deficient (Airewt/mut ) mTECs cocultured with WT single-positive (SP) CD4+ thymocytes. Although the libraries differed in their mRNA and long noncoding RNA (lncRNA) profiles, indicating that mTECs were heterogeneous in terms of their transcriptome, UMAP clustering revealed that both mTEC lines expressed their specific markers, i.e., Epcam, Itgb4, Itga6, and Casp3 in resting mTECs and Ccna2, Pbk, and Birc5 in proliferative mTECs. Both cocultured SP CD4+ thymocytes remained in a homogeneous cluster expressing the Il7r and Ccr7 markers. Comparisons of the two types of cocultures revealed the differential expression of mRNAs that encode transcription factors (Zfpm2, Satb1, and Lef1), cell adhesion genes (Itgb1) in mTECs, and Themis in thymocytes, which is associated with the regulation of positive and negative selection. At the single-cell sequencing resolution, we observed that Aire acts on both Aire WT and Aire-deficient mTECs as an upstream controller of mRNAs, which encode transcription factors or adhesion proteins that, in turn, are posttranscriptionally controlled by lncRNAs, for example, Neat1, Malat1, Pvt1, and Dancr among others. Under Aire deficiency, mTECs dysregulate the expression of MHC-II, CD80, and CD326 (EPCAM) protein markers as well as metabolism and cell cycle-related mRNAs, which delay the cell cycle progression. Moreover, when adhered to mTECs, WT SP CD4+ or CD8+ thymocytes modulate the expression of cell activation proteins, including CD28 and CD152/CTLA4, and the expression of cellular metabolism mRNAs. These findings indicate a complex mechanism through which an imbalance in Aire expression can affect mTECs and thymocytes during adhesion.
Assuntos
Proteína AIRE , Adesão Celular , Células Epiteliais , RNA Longo não Codificante , Timócitos , Fatores de Transcrição , Transcriptoma , RNA Longo não Codificante/genética , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Camundongos , Timócitos/metabolismo , Timócitos/imunologia , Timócitos/citologia , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismo , Análise de Célula Única , Redes Reguladoras de Genes , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Perfilação da Expressão Gênica , Camundongos KnockoutRESUMO
BACKGROUND: Capsaicin, a bioactive compound found in peppers, is recognized for its anti-inflammatory, antioxidant, and anti-lipidemic properties. This study aimed to evaluate the effects of capsaicin on atherosclerosis progression. METHODS: Apolipoprotein E knockout mice and their C57BL/6 controls were utilized to assess blood lipid profile, inflammatory status, and atherosclerotic lesions. We also examined the influence of capsaicin on cholesterol influx and efflux, and the role of TRPV1 and PPARγ signaling pathways in bone marrow-derived macrophages. RESULTS: Capsaicin treatment reduced weight gain, visceral adiposity, blood triglycerides, and total and non-HDL cholesterol. These improvements were associated with a reduction in atherosclerotic lesions in the aorta and carotid. Capsaicin also improved hepatic oxidative and inflammatory status. Systemic inflammation was also reduced, as indicated by reduced leukocyte rolling and adhesion on the mesenteric plexus. Capsaicin decreased foam cell formation by reducing cholesterol influx through scavenger receptor A and increasing cholesterol efflux via ATP-binding cassette transporter A1, an effect primarily linked to TRPV1 activation. CONCLUSIONS: These findings underscore the potential of capsaicin as a promising agent for atherosclerosis prevention, highlighting its comprehensive role in modulating lipid metabolism, foam cell formation, and inflammatory responses.
Assuntos
Aterosclerose , Capsaicina , Células Espumosas , Inflamação , PPAR gama , Canais de Cátion TRPV , Animais , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Aterosclerose/prevenção & controle , Aterosclerose/tratamento farmacológico , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Capsaicina/farmacologia , Colesterol/sangue , Colesterol/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Inflamação/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Knockout para ApoE , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismoRESUMO
The two-kidney, one-clip (2K1C) Goldblatt rodent model elicits a reduction in renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio-ability causes the accumulation of the tricarboxylic cycle intermediary, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, OXGR1 is abundantly expressed in intercalated cells (ICs) of the collecting duct (CD), thus contributing to sodium transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin system (RAS), is a key regulator of sodium reabsorption and blood pressure (BP) that is expressed in ICs. The PRR is upregulated in 2K1C rats. Here, we tested the hypothesis that chronic reduction in RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alters sodium balance and BP in 2K1C mice. To determine the role of OXGR1 in regulating the PRR in the CDs during renovascular hypertension, we performed 2K1C Goldblatt surgery (clip = 0.13 mm internal gap, 14 days) in two groups of male mice: (1) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day); (2) OXGR1-/- knockout mice. Wild-type and sham-operated mice were used as controls. After 14 days, 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p < 0.01) and a lower natriuretic response after the saline challenge test. The CK group showed upregulation of erythropoietin, augmented α-ketoglutarate, and increased PRR expression in the renal medulla. The CK of OXGR1 knockout mice and mice subjected to the OXGR1 antagonist elicited impaired PRR upregulation, attenuated SBP, and better natriuretic responses. In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.
Assuntos
Túbulos Renais Coletores , Receptores de Superfície Celular , Sódio , Regulação para Cima , Animais , Camundongos , Túbulos Renais Coletores/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Masculino , Sódio/metabolismo , Hipertensão Renovascular/metabolismo , Hipertensão Renovascular/genética , Pressão Sanguínea , Camundongos Knockout , Receptor de Pró-Renina , Rim/metabolismo , Modelos Animais de Doenças , Sistema Renina-Angiotensina , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Purinérgicos P2RESUMO
Dietary sodium restriction increases plasma triglycerides (TG) and total cholesterol (TC) concentrations as well as causing insulin resistance and stimulation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system. Stimulation of the angiotensin II type-1 receptor (AT1) is associated with insulin resistance, inflammation, and the inhibition of adipogenesis. The current study investigated whether aerobic exercise training (AET) mitigates or inhibits the adverse effects of dietary sodium restriction on adiposity, inflammation, and insulin sensitivity in periepididymal adipose tissue. LDL receptor knockout mice were fed either a normal-sodium (NS; 1.27% NaCl) or a low-sodium (LS; 0.15% NaCl) diet and were either subjected to AET for 90 days or kept sedentary. Body mass, blood pressure (BP), hematocrit, plasma TC, TG, glucose and 24-hour urinary sodium (UNa) concentrations, insulin sensitivity, lipoprotein profile, histopathological analyses, and gene and protein expression were determined. The results were evaluated using two-way ANOVA. Differences were not observed in BP, hematocrit, diet consumption, and TC. The LS diet was found to enhance body mass, insulin resistance, plasma glucose, TG, LDL-C, and VLDL-TG and reduce UNa, HDL-C, and HDL-TG, showing a pro-atherogenic lipid profile. In periepididymal adipose tissue, the LS diet increased tissue mass, TG, TC, AT1 receptor, pro-inflammatory macro-phages contents, and the area of adipocytes; contrarily, the LS diet decreased anti-inflammatory macrophages, protein contents and the transcription of genes related to insulin sensitivity. The AET prevented insulin resistance, but did not protect against dyslipidemia, adipose tissue pro-inflammatory profile, increased tissue mass, AT1 receptor expression, TG, and TC induced by the LS diet.
Assuntos
Adiposidade , Dieta Hipossódica , Inflamação , Resistência à Insulina , Condicionamento Físico Animal , Animais , Camundongos , Inflamação/metabolismo , Inflamação/prevenção & controle , Masculino , Camundongos Knockout , Gordura Intra-Abdominal/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
Toxoplasmosis is a globally significant disease that poses a severe threat to immunocompromised individuals, especially in Brazil, where a high prevalence of virulent and atypical strains of Toxoplasma gondii is observed. In 1998, the EGS strain, exhibiting a unique infection phenotype, was isolated in Brazil, adding to the complexity of strain diversity. The P2X7 receptor is critical in inflammation and controlling intracellular microorganisms such as T. gondii. However, its genetic variability can result in receptor dysfunction, potentially worsening susceptibility. This study investigates the role of the P2X7 receptor during acute infection induced by the EGS atypical strain, offering insight into the mechanisms of T. gondii infection in this context. We infected the female C57BL/6 (WT) or P2X7 knockout (P2X7-/-) by gavage. The EGS infection causes intestinal inflammation. The P2X7-/- mice presented higher parasite load in the intestine, spleen, and liver. The absence of the P2X7 receptor disrupts inflammatory cell balance by reducing NLRP3, IL-1ß, and Foxp3 expression while increasing IFN-γ expression and production in the intestine. In the liver, P2X7-/- animals demonstrate diminished inflammatory infiltrate within the portal and lobular regions concurrent with an enlargement of the spleen. In conclusion, the infection of mice with the EGS strain elicited immune alterations, leading to acute inflammation and cytokine dysregulation, while the P2X7 receptor conferred protection against parasitic proliferation across multiple organs.