RESUMEN
ABSTRACT: Previous studies have found that anxiety disorders may increase the incidence of atrial fibrillation (AF). More and more studies have shown that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are involved in the occurrence and development of cardiovascular diseases. However, the role of AMPARs in AF associated with anxiety disorder remains unclear. The aim of this study was to investigate the effect of AMPARs on AF susceptibility in rats with anxiety disorder and its possible mechanism. The anxiety disorder rat model was established by unpredictable empty bottle stimulation and was treated with AMPARs agonist and antagonist. Our results showed that AMPARs antagonist treatment significantly reduced sympathetic activity, improved heart rate variability, shortened action potential duration, prolonged effective refractory period, reduced AF induction rate, and improved cardiac electrical remodeling and the expression of inflammatory factors. In addition, inhibition of AMPARs reduced the phosphorylation of IκBα and p65. Our experimental results suggest that inhibition of AMPARs can reduce autonomic remodeling, improve atrial electrical remodeling, and suppress myocardial inflammation, which provides a potential therapeutic strategy for the treatment of AF associated with anxiety disorder.
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Trastornos de Ansiedad , Fibrilación Atrial , Modelos Animales de Enfermedad , Atrios Cardíacos , Ratas Sprague-Dawley , Receptores AMPA , Animales , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/metabolismo , Masculino , Trastornos de Ansiedad/tratamiento farmacológico , Trastornos de Ansiedad/metabolismo , Trastornos de Ansiedad/fisiopatología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Receptores AMPA/metabolismo , Remodelación Atrial/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Potenciales de Acción/efectos de los fármacos , Fosforilación , Transducción de Señal , Sistema Nervioso Simpático/fisiopatología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Factor de Transcripción ReIA/metabolismo , Ratas , Antiinflamatorios/farmacología , Periodo Refractario Electrofisiológico/efectos de los fármacos , Inhibidor NF-kappaB alfa/metabolismoRESUMEN
In osteoarthritis (OA), extracellular matrix (ECM) digestion by cartilage-degrading enzymes drives cartilage destruction and generates ECM fragments, such as proteoglycan aggrecan (PG) peptides. PG peptides have been shown to induce immunological functions of chondrocytes. However, the role of PG peptides in stimulating catabolic mediators from chondrocytes has not been investigated. Therefore, we aim to determine the effects and its mechanism by which PG peptides induce chondrocytes to produce catabolic mediators in OA. Human chondrocytes were stimulated with IFNγ and various PG peptides either (i) with or (ii) without TLR2 blockade or (iii) with Lactobacillus species-conditioned medium (LCM), a genus of bacteria with anti-inflammatory properties. Transcriptomic analysis, cartilage-degrading enzyme production and TLR2-intracellular signaling activation were investigated. Chondrocytes treated with PG peptides p16-31 and p263-280 increased expression levels of genes associated with chondrocyte hypertrophy, cartilage degradation and proteolytic enzyme production. TLR2 downstream signaling proteins (STAT3, IkBα and MAPK9) were significantly phosphorylated in p263-280 peptide-stimulated chondrocytes. MMP-1 and ADAMTS-4 were significantly reduced in p263-280 peptides-treated condition with TLR2 blockade or LCM treatment. Phosphorylation levels of IkBa, ERK1/2 and MAPK9 were significantly decreased with TLR2 blockade, but only phosphorylation levels of MAPK9 was significantly decreased with LCM treatment. Our study showed that PG peptide stimulation via TLR2 induced cartilage-degrading enzyme production via activation of MAPK, NFκB and STAT3 pathways.
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Agrecanos , Condrocitos , Lactobacillus , Receptor Toll-Like 2 , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Humanos , Receptor Toll-Like 2/metabolismo , Agrecanos/metabolismo , Medios de Cultivo Condicionados/farmacología , Lactobacillus/metabolismo , Transducción de Señal/efectos de los fármacos , Osteoartritis/metabolismo , Osteoartritis/patología , Células Cultivadas , Proteína ADAMTS4/metabolismo , Factor de Transcripción STAT3/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Inhibidor NF-kappaB alfa/metabolismoRESUMEN
Background: Osteoporosis is a disease associated with bone resorption, characterized primarily by the excessive activation of osteoclasts. Ginkgetin is a compound purified from natural ginkgo leaves which has various biological properties, including anti-inflammation, antioxidant, and anti-tumor effects. This study investigated the bone-protective effects of ginkgetin in ovariectomized (OVX) mice and explored their potential signaling pathway in inhibiting osteoclastogenesis in a mouse model of osteoporosis. Methods: Biochemical assays were performed to assess the levels of Ca, ALP, and P in the blood. Micro CT scanning was used to evaluate the impact of ginkgetin on bone loss in mice. RT-PCR was employed to detect the expression of osteoclast-related genes (ctsk, c-fos, trap) in their femoral tissue. Hematoxylin and eosin (H&E) staining was utilized to assess the histopathological changes in femoral tissue due to ginkgetin. The TRAP staining was used to evaluate the impact of ginkgetin osteoclast generation in vivo. Western blot analysis was conducted to investigate the effect of ginkgetin on the expression of p-NF-κB p65 and IκBα proteins in mice. Results: Our findings indicate that ginkgetin may increase the serum levels of ALP and P, while decreasing the serum level of Ca in OVX mice. H&E staining and micro CT scanning results suggest that ginkgetin can inhibit bone loss in OVX mice. The TRAP staining results showed ginkgetin suppresses the generation of osteoclasts in OVX mice. RT-PCR results demonstrate that ginkgetin downregulate the expression of osteoclast-related genes (ctsk, c-fos, trap) in the femoral tissue of mice, and this effect is dose-dependent. Western blot analysis results reveal that ginkgetin can inhibit the expression of p-NF-κB p65 and IκBα proteins in mice. Conclusion: Ginkgetin can impact osteoclast formation and activation in OVX mice by inhibiting the NF-κB/IκBα signaling pathway, thereby attenuating bone loss in mice.
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Biflavonoides , FN-kappa B , Osteoclastos , Transducción de Señal , Animales , Biflavonoides/farmacología , Biflavonoides/uso terapéutico , Transducción de Señal/efectos de los fármacos , Ratones , FN-kappa B/metabolismo , Femenino , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/patología , Ovariectomía , Modelos Animales de Enfermedad , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/prevención & control , Resorción Ósea/patología , Microtomografía por Rayos X , Inhibidor NF-kappaB alfa/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: We aimed to investigate the serum Nuclear Factor Kappa B (NF-κB) p105, NF-κB p65 and Inhibitor Kappa B Alpha (IκBα) levels in patients with mild/moderate Coronavirus Disease 2019 (COVID-19) and their association with the course of the disease. MATERIALS AND METHODS: Blood was drawn from 35 COVID-19 patients who applied to the Department of Emergency Medicine of Istanbul University-Cerrahpasa at the time of diagnosis and from 35 healthy individuals. The patients were evaluated to have mild/moderate degree of disease according to National Early Warning Score 2 (NEWS2) scoring and computed tomography (CT) findings. The markers were studied in the obtained serum samples, using enzyme-linked immunoassay (ELISA). Receiver Operating Characteristic (ROC) analysis was performed. Statistical significance was evaluated to be p < 0.05. RESULTS: NF-κB p105 levels were significantly higher in the COVID-19 group compared to the control group. C-reactive protein (CRP), D-dimer, ferritin levels of the patients were significantly higher (p < 0.001) compared to the control group, while the lymphocyte count was found lower (p = 0.001). IκBα and NF-κB p65 levels are similar in both groups. Threshold value for NF-κB p105 was above 0.78 ng/mL, sensitivity was 71.4% and specificity was 97.1% (p < 0.05). NF-κB p105 levels at the time of diagnosis of the patients who required supplemental oxygen (O2), were significantly higher (p < 0.01). CONCLUSIONS: The rise in serum NF-κB p105 levels during the early stages of infection holds diagnostic value. Besides its relation with severity might have a prognostic feature to foresee the requirement for supplemental O2 that occurs during hospitalization.
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Biomarcadores , Proteína C-Reactiva , COVID-19 , SARS-CoV-2 , Humanos , COVID-19/sangre , COVID-19/diagnóstico , Masculino , Femenino , Persona de Mediana Edad , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Adulto , Inhibidor NF-kappaB alfa/metabolismo , Factor de Transcripción ReIA/metabolismo , Anciano , Subunidad p50 de NF-kappa B/metabolismo , FN-kappa B/metabolismo , Curva ROC , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Ferritinas/sangreRESUMEN
Neuroinflammation is a prominent feature of Alzheimer's disease (AD). Activated microglia undergo a reprogramming of cellular metabolism necessary to power their cellular activities during disease. Thus, selective targeting of microglial immunometabolism might be of therapeutic benefit for treating AD. In the AD brain, the levels of microglial hexokinase 2 (HK2), an enzyme that supports inflammatory responses by promoting glycolysis, are significantly increased. In addition, HK2 displays non-metabolic activities that extend its inflammatory role beyond glycolysis. The antagonism of HK2 affects microglial phenotypes and disease progression in a gene-dose-dependent manner. HK2 complete loss fails to improve pathology by exacerbating inflammation, while its haploinsufficiency reduces pathology in 5xFAD mice. We propose that the partial antagonism of HK2 is effective in slowing disease progression by modulating NF-κB signaling through its cytosolic target, IKBα. The complete loss of HK2 affects additional inflammatory mechanisms related to mitochondrial dysfunction.
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Enfermedad de Alzheimer , Progresión de la Enfermedad , Hexoquinasa , Microglía , Hexoquinasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Animales , Microglía/metabolismo , Microglía/efectos de los fármacos , Microglía/patología , Ratones , Humanos , FN-kappa B/metabolismo , Ratones Transgénicos , Transducción de Señal , Inhibidor NF-kappaB alfa/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Inflamación/patología , Inflamación/metabolismo , Encéfalo/patología , Encéfalo/metabolismo , Glucólisis/efectos de los fármacos , Dosificación de GenRESUMEN
Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.
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Infecciones por Adenoviridae , Fibroblastos , FN-kappa B , Fosfatidilinositol 3-Quinasas , Enfermedades de las Aves de Corral , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Fibroblastos/virología , Embrión de Pollo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Infecciones por Adenoviridae/veterinaria , Infecciones por Adenoviridae/virología , Infecciones por Adenoviridae/inmunología , Enfermedades de las Aves de Corral/virología , Inflamación , Aviadenovirus/fisiología , Citocinas/metabolismo , Pollos , Serogrupo , Inhibidor NF-kappaB alfa/metabolismoRESUMEN
Anisomeles indica (L.) Kuntze is a traditional herb with multiple medicinal properties and with potential for preventing or treating various diseases. Acteoside, one of the active ingredients in A. indica, is prepared into commercially available products of A. indica HP813 powder. In this study, the gastroprotective effects of A. indica HP813 powder were evaluated. Wistar rats were treated with A. indica HP813 powder at doses of 0, 207.5, 415, and 830 mg/kg body weight for 28 days. Then, gastric ulcers were induced by the oral administration of 70% ethanol (10 mL/kg body weight) on day 28. The rats were sacrificed at the end of the trial, and stomach tissues were collected. These stomach tissues were then used for macroscopic, microscopic, and immunohistochemical analyses. The results indicated that the area of gastric ulcer was 48.61%, 35.30%, and 27.16% in the ethanol-induced group, 415 mg/kg A. indica HP813 powder group, and 830 mg/kg A. indica HP813 powder group, respectively. In addition, the lesion scores were 2.9, 2.4, and 2.3 in the ethanol-induced group, 415 mg/kg A. indica HP813 powder group, and 830 mg/kg A. indica HP813 powder group, respectively. The immunochemical staining of the gastric tissue revealed that A. indica HP813 powder reduced the expressions of TNF-α and NF-κB proteins in the gastric tissue, which had been induced by ethanol. Finally, A. indica HP813 powder protected the gastric ulcer from ethanol damage through IκB-α induction. The present results demonstrated that A. indica HP813 powder has protective effects against ethanol-induced gastric ulcer.
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Antiulcerosos , Etanol , Inhibidor NF-kappaB alfa , FN-kappa B , Úlcera Gástrica , Animales , Masculino , Ratas , Antiulcerosos/farmacología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Extractos Vegetales/farmacología , Polvos , Ratas Wistar , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/prevención & control , Úlcera Gástrica/tratamiento farmacológico , Úlcera Gástrica/metabolismoRESUMEN
As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Inhibidor NF-kappaB alfa , Organoides , SARS-CoV-2 , Replicación Viral , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , COVID-19/virología , COVID-19/metabolismo , COVID-19/genética , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Organoides/virología , Organoides/metabolismo , SARS-CoV-2/fisiologíaRESUMEN
BACKGROUND: Oncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge. METHODS: In this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082. RESULTS: We demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition. CONCLUSION: Our study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers.
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Antineoplásicos , Nitrilos , Sulfonas , Humanos , Nitrilos/farmacología , Sulfonas/farmacología , Animales , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Mutación/genética , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Proteínas ras/metabolismo , Proteínas ras/antagonistas & inhibidoresRESUMEN
Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.
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Proliferación Celular , Células Madre Hematopoyéticas , Inhibidor NF-kappaB alfa , Transducción de Señal , Tretinoina , Animales , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Tretinoina/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Ratones , Desarrollo Embrionario/genética , Ratones Noqueados , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/genética , Ratones Endogámicos C57BL , Regulación del Desarrollo de la Expresión Génica , FemeninoRESUMEN
Inflammation-mediated dysfunction of cardiomyocytes is the main cause of diabetic cardiomyopathy (DCM). The present study aimed to investigate the roles of siah E3 ubiquitin protein ligase 1 (SIAH1) in DCM. The online dataset GSE4172 was used to analyze the differentially expressed genes in myocardial inflammation of DCM patients. RT-qPCR was conducted to detect mRNA levels. Enzyme-Linked Immunosorbent Assay (ELISA) was performed to detect cytokine release. Western blot was used to detect protein expression. Lactate dehydrogenase (LDH) assay was used to determine cytotoxicity. In vitro ubiquitination assay was applied to determine the ubiquitination of nuclear factor kappa B inhibitor alpha (1κÐ-α). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cardiomyocytes. Flow cytometry was applied for determining cardiomyocyte pyroptosis. The results showed that SIAH1 was overexpressed in human inflammatory cardiomyopathy. High expression of SIAH1 was associated with inflammatory response. SIAH1 was also overexpressed lipopolysaccharide (LPS)-induced inflammatory cardiomyopathy model in vitro. However, SIAH1 knockdown suppressed the inflammatory-related pyroptosis of cardiomyocytes. SIAH1 promoted the ubiquitination of 1κÐ-α and activated nuclear factor kappa Ð (NF-κÐ) signaling, which promoted the pyroptosis of cardiomyocytes. In conclusion, SIAH1 exacerbated the progression of human inflammatory cardiomyopathy via inducing the ubiquitination of 1κÐ-α and activation of NF-κÐ signaling. Therefore, SIAHI/IκB-α/NF-κB signaling may be a potential target for human inflammatory cardiomyopathy.
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Cardiomiopatías Diabéticas , Miocitos Cardíacos , FN-kappa B , Piroptosis , Transducción de Señal , Ubiquitina-Proteína Ligasas , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Humanos , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Ubiquitinación , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/genéticaRESUMEN
Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop lifelong neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pretreated with SCFA cocktail before JEV infection. Cytokine bead analysis, immunoblotting, and PCR were performed to analyze relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq, and bioinformatics tools were used for differentially expressed (DE) miRNAs and weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in monocyte chemoattractant protein (MCP1) and tumor necrosis factor alpha (TNFα) along with reduced expression of phospho-nuclear factor kappa B (phospho-NF-κB) was observed in SCFA conditions. Significant attenuation of histone deacetylase activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA + JEV-treated cells at 6 h post-infection. WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR-200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκßα that possibly dampened NF-κB signal activation downstream. IMPORTANCE: The gut-brain axis plays a pivotal role in the physiological state of an organism. Gut microbiota-derived metabolites are known to play a role in brain disorders including neuroviral infections. Short-chain fatty acids (SCFAs) appear to quench inflammatory markers in Japanese encephalitis virus-infected microglial cells in vitro. Mechanistically, we demonstrate the interaction between miR-200a-3p and ZBTB20 in regulating the canonical nuclear factor kappa B (NF-κB) signaling pathway via transcriptional regulation of Iκßα. Findings of this study pave the way to a better understanding of SCFA mechanisms that can be used to develop strategies against viral neuroinflammation.
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Virus de la Encefalitis Japonesa (Especie) , Ácidos Grasos Volátiles , Inflamación , MicroARNs , Microglía , MicroARNs/genética , MicroARNs/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/genética , Microglía/virología , Microglía/metabolismo , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/farmacología , Ratones , Animales , Inflamación/genética , Inflamación/virología , Encefalitis Japonesa/virología , Encefalitis Japonesa/genética , Encefalitis Japonesa/metabolismo , Línea Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal , HumanosRESUMEN
Cadmium (Cd) exposure is considered as non-infectious stressor to human and animal health. Recent studies suggest that the immunotoxicity of low dose Cd is not directly apparent, but disrupts the immune responses when infected with some bacteria or virus. But how Cd alters the adaptive immunity organ and cells remains unclear. In this study, we applied lipopolysaccharide (LPS, infectious stressor) to induced inflammation in spleen tissues and T cells, and investigated the effects after Cd exposure and the underlying mechanism. Cd exposure promoted LPS-induced the expressions of the inflammatory factors, induced abnormal initiation of autophagy, but blocked autophagic flux. The effects Cd exposure under LPS activation were reversed by the autophagy promoter Rapamycin. Under LPS activation conditions, Cd also induced oxidative stress by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and reducing total antioxidant capacity (T-AOC) activity. The increased superoxide dismutase (SOD) activity after Cd exposure might be a negative feedback or passive adaptive regulation of oxidative stress. Cd-increased autophagic flux inhibition and TNF-α expression were reversed by ROS scavenger α-tocopherol (TCP). Furthermore, under LPS activation condition, Cd promoted activation of toll-like receptor 4 (TLR4)/IκBα/NFκ-B signaling pathway and increased TLR4 protein stability, which were abolished by the pretreatment of Rapamycin. The present study confirmed that, by increasing ROS-mediated inhibiting autophagic degradation of TLR4, Cd promoted LPS-induced inflammation in spleen T cells. This study identified the mechanism of autophagy in Cd-aggravated immunotoxicity under infectious stress, which could arouse public attention to synergistic toxicity of Cd and bacterial or virus infection.
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Autofagia , Cadmio , Inflamación , Lipopolisacáridos , FN-kappa B , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de Señal , Receptor Toll-Like 4 , Cadmio/toxicidad , Autofagia/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Inflamación/inducido químicamente , Estrés Oxidativo/efectos de los fármacos , Ratones , Bazo/efectos de los fármacos , Inhibidor NF-kappaB alfa/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , MasculinoRESUMEN
Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.
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Citometría de Flujo , Inhibidor NF-kappaB alfa , FN-kappa B , Humanos , Citometría de Flujo/métodos , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Proteolisis , Células THP-1 , Macrófagos/metabolismo , Macrófagos/inmunologíaRESUMEN
MicroRNAs (miRNAs) are a crucial type of non-coding RNAs involved in post-transcriptional regulation. The playing essential regulatory roles in the NF-κB signaling pathway and modulate the host immune response to diverse pathogens by targeting IκBα. However, the regulatory mechanism of miRNAs in relation with IκBα in Sebastes schlegelii remains unclear. In our study, we identified two copies of IkBα gene in black rockfish (Sebastes schlegelii), namely IkBα1 and IkBα2. Moreover, we have discovered that miRNA-530 can activate the NF-κB signaling pathway by inhibiting the expression of IκBα, thereby inducing the inflammatory response. This project comprehensively investigated the interactive regulatory roles of miRNA-530 in the NF-κB signaling pathway at both cellular and in vivo levels, while also elucidating the regulatory relationships between miRNA-530 and IκBα. In conclusion, our research confirmed that miRNA-530 can target the 3'UTR region of IκBα, resulting in a decrease in the expression of IκBα at the post-transcriptional level and inhibiting its translation. The findings contribute to the understanding of the regulatory network of non-coding RNA in teleosts and its subsequent regulation of the NF-κB signaling pathway by miRNAs.
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Regulación de la Expresión Génica , MicroARNs , Inhibidor NF-kappaB alfa , FN-kappa B , Transducción de Señal , MicroARNs/genética , MicroARNs/metabolismo , Animales , FN-kappa B/genética , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Regulación de la Expresión Génica/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Inmunidad Innata/genética , Peces/genética , Peces/inmunología , Perciformes/genética , Perciformes/inmunologíaRESUMEN
BACKGROUND: While de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood. METHODS: We conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses. RESULTS: Mechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy. CONCLUSIONS: Thus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.
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Neoplasias Colorrectales , Resistencia a Antineoplásicos , Fluorouracilo , FN-kappa B , Escualeno-Monooxigenasa , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Humanos , Escualeno-Monooxigenasa/metabolismo , Escualeno-Monooxigenasa/genética , FN-kappa B/metabolismo , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Ratones , Línea Celular Tumoral , Ratones Desnudos , Ratones Endogámicos BALB C , Femenino , Masculino , Proliferación Celular/efectos de los fármacos , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic inflammatory pain is the highest priority for people with osteoarthritis when seeking medical attention. Despite the availability of NSAIDs and glucocorticoids, central sensitization and peripheral sensitization make pain increasingly difficult to control. Previous studies have identified the ubiquitination system as an important role in the chronic inflammatory pain. Our study displayed that the E3 ubiquitin ligase tripartite motif-containing 14 (Trim14) was abnormally elevated in the serum of patients with osteoarthritis and pain, and the degree of pain was positively correlated with the degree of Trim14 elevation. Furthermore, CFA-induced inflammatory pain rat model showed that Trim14 was significantly increased in the L3-5 spinal dorsal horn (SDH) and dorsal root ganglion (DRG), and in turn the inhibitor of nuclear factor Kappa-B isoform α (IκBα) was decreased after Trim14 elevation. After intrathecal injection of Trim14 siRNA to inhibit Trim14 expression, IκBα expression was reversed and increased, and the pain behaviors and anxiety behaviors of rats were significantly relieved. Overall, these findings suggested that Trim14 may contribute to chronic inflammatory pain by degrading IκBα, and that Trim14 may become a novel therapeutic target for chronic inflammatory pain.
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Dolor Crónico , Inflamación , Inhibidor NF-kappaB alfa , Osteoartritis , Ratas Sprague-Dawley , Transducción de Señal , Anciano , Animales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Dolor Crónico/metabolismo , Ganglios Espinales/metabolismo , Inflamación/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Osteoartritis/metabolismo , Transducción de Señal/fisiología , Asta Dorsal de la Médula Espinal/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8') of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (-)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1ß and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1ß and TNF-α production by inhibiting IKBα/p65 pathway activation.
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Relación Dosis-Respuesta a Droga , Raíces de Plantas , Sesquiterpenos , Transducción de Señal , Sesquiterpenos/farmacología , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificación , Raíces de Plantas/química , Transducción de Señal/efectos de los fármacos , Estructura Molecular , Ratones , Animales , Relación Estructura-Actividad , Factor de Transcripción ReIA/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Descubrimiento de Drogas , Inhibidor NF-kappaB alfa/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificaciónRESUMEN
Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.
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Citocinas , Quinasa I-kappa B , Inhibidor NF-kappaB alfa , Humanos , Quinasa I-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Inhibidor NF-kappaB alfa/metabolismo , Citocinas/metabolismo , Eritromicina/farmacología , Eritromicina/química , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Macrólidos/farmacología , Macrólidos/química , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitinâproteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.