RESUMO
BACKGROUND/AIMS: Adrenaline quickly inhibits the release of histamine from mast cells. Besides ß2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. RESULTS: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a ß2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells. CONCLUSION: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by ß2-adrenergic receptors.
Assuntos
Degranulação Celular , Epinefrina , Exocitose , Mastócitos , Prazosina , Animais , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/citologia , Epinefrina/farmacologia , Ratos , Prazosina/farmacologia , Degranulação Celular/efeitos dos fármacos , Masculino , Exocitose/efeitos dos fármacos , Técnicas de Patch-Clamp , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Ratos WistarRESUMO
Sympathetic hyperinnervation is the leading cause of fatal ventricular arrhythmia (VA) after myocardial infarction (MI). Cardiac mast cells cause arrhythmias directly through degranulation. However, the role and mechanism of mast cell degranulation in sympathetic remodeling remain unknown. We investigated the role of oxytocin (OT) in stabilizing cardiac mast cells and improving sympathetic innervation in rats. MI was induced by coronary artery ligation. Western blotting, immunofluorescence, and toluidine staining of mast cells were performed to determine the expression and location of target protein. Mast cells accumulated significantly in peri-infarcted tissues and were present in a degranulated state. They expressed OT receptor (OTR), and OT infusion reduced the number of degranulated cardiac mast cells post-MI. Sympathetic hyperinnervation was attenuated as assessed by immunofluorescence for tyrosine hydroxylase (TH). Seven days post-MI, the arrhythmia score of programmed electrical stimulation was higher in vehicle-treated rats with MI than in rats treated with OT. An in vitro study showed that OT stabilized mast cells via the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Further in vivo studies on OTR-deficient mice showed worsening mast cell degranulation and worsening sympathetic innervation. OT pretreatment inhibited cardiac mast cell degranulation post-MI and prevented sympathetic hyperinnervation, along with mast cell stabilization via the PI3K/Akt pathway. SIGNIFICANCE STATEMENT: This is the first study to elucidate the role and mechanism of oxytocin (OT) in inflammatory-sympathetic communication mediated sympathetic hyperinnervation after myocardial infarction (MI), providing new approaches to prevent fatal arrhythmias.
Assuntos
Degranulação Celular , Mastócitos , Infarto do Miocárdio , Ocitocina , Ratos Sprague-Dawley , Receptores de Ocitocina , Sistema Nervoso Simpático , Animais , Ocitocina/farmacologia , Ocitocina/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ratos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Masculino , Degranulação Celular/efeitos dos fármacos , Receptores de Ocitocina/metabolismo , Receptores de Ocitocina/antagonistas & inibidores , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologiaRESUMO
Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement.
Assuntos
Ácido Betulínico , Degranulação Celular , Membrana Celular , Mastócitos , Triterpenos Pentacíclicos , Receptores de IgE , Triterpenos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Triterpenos Pentacíclicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Receptores de IgE/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Ratos , Fosforilação/efeitos dos fármacos , Citocinas/metabolismoRESUMO
Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients' blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients' clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients' blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive-(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.
Assuntos
Citocinas , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/tratamento farmacológico , Pessoa de Meia-Idade , Masculino , Adulto , Feminino , Citocinas/metabolismo , Idoso , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Degranulação Celular/efeitos dos fármacos , Adulto JovemRESUMO
Marrubiin is a diterpene with a long history of a wide range of biological activities. In this study, the anti-inflammatory effects of marrubiin were investigated using several in vitro and in vivo assays. Marrubiin inhibited carrageenan-induced peritoneal inflammation by preventing inflammatory cell infiltration and peritoneal mast cell degranulation. The anti-inflammatory activity was further demonstrated by monitoring a set of biochemical parameters, showing that the peritoneal fluid of animals treated with marrubiin had lower levels of proteins and lower myeloperoxidase activity compared with the fluid of animals that were not treated. Marrubiin exerted the most pronounced cytotoxic activity towards peripheral mononuclear cells, being the main contributors to peritoneal inflammation. Additionally, a moderate lipoxygenase inhibition activity of marrubiin was observed.
Assuntos
Anti-Inflamatórios , Carragenina , Diterpenos , Mastócitos , Animais , Carragenina/efeitos adversos , Camundongos , Diterpenos/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Anti-Inflamatórios/farmacologia , Camundongos Endogâmicos C57BL , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Peritonite/patologia , Masculino , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/patologia , Degranulação Celular/efeitos dos fármacos , Peroxidase/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismoRESUMO
Inhibitors of sodium/glucose cotransporter 2 (SGLT2), such as empagliflozin and canagliflozin, have been widely used to block glucose reabsorption in the proximal tubules of kidneys in patients with diabetes. A meta-analysis suggested that SGLT2 inhibitors are associated with a decreased risk of asthma development. Therefore, we investigated whether SGLT2 inhibitors could suppress allergic asthma. Empagliflozin and canagliflozin suppressed the in vitro degranulation reaction induced by antigens in a concentration-dependent manner in RBL-2H3 mast cells. Empagliflozin and canagliflozin were administered to BALB/c mice sensitized to ovalbumin (OVA). The administration of empagliflozin or canagliflozin significantly suppressed OVA-induced airway hyper-responsiveness and increased the number of immune cells and pro-inflammatory cytokine mRNA expression levels in bronchoalveolar lavage fluid. The administration of empagliflozin and canagliflozin also suppressed OVA-induced histopathological changes in the lungs. Empagliflozin and canagliflozin also suppressed serum IgE levels. These results suggested that empagliflozin and canagliflozin may be applicable for the treatment of allergic asthma by suppressing immune responses.
Assuntos
Asma , Compostos Benzidrílicos , Canagliflozina , Glucosídeos , Camundongos Endogâmicos BALB C , Ovalbumina , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Asma/tratamento farmacológico , Asma/imunologia , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Camundongos , Ovalbumina/efeitos adversos , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Líquido da Lavagem Broncoalveolar , Ratos , Citocinas/metabolismo , Degranulação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Transportador 2 de Glucose-Sódio/metabolismo , MasculinoRESUMO
BACKGROUND: Food allergy has become a global public health problem. This study aimed to explore the possible anti-allergic effect of vitamin C (VC). A rat basophilic leukemia (RBL)-2H3 cell degranulation model was used to assess the effect of VC on degranulation in vitro, and an ovalbumin (OVA)-induced BALB/c mouse allergy model was used to assess the anti-allergy effect of VC in vivo. RESULTS: In vitro, VC significantly attenuated the release of ß-hexosaminidase, tryptase and histamine, and also reduced cytokine production (interleukins 4 and 6, tumor necrosis factor α) significantly (P < 0.05), with the inhibitory effect demonstrating a positive correlation with VC dose. In vivo, compared with the OVA group, the levels of serum immunoglobulins E and G1 of the VC low-dose (VCL) group (50 mg kg-1) and high-dose (VCH) group (200 mg·kg-1) were significantly reduced (P < 0.05). Furthermore, the plasma histamine level was also significantly decreased (P < 0.05). Moreover, TH2 cell polarization in mice of the VCL and VCH groups was significantly inhibited (P < 0.05), promoting the TH1/TH2 cell polarization balance. Additionally, VC treatment enhanced the expression of CD80 (P < 0.05) in spleen and small intestine tissues, while significantly inhibiting the expression of CD86 (P < 0.05); notably, high-dose VC treatment was more effective. CONCLUSION: VC exerted an anti-allergic effect through inhibiting degranulation and regulating TH1/TH2 cell polarization balance. © 2024 Society of Chemical Industry.
Assuntos
Antialérgicos , Ácido Ascórbico , Degranulação Celular , Hipersensibilidade Alimentar , Camundongos Endogâmicos BALB C , Células Th1 , Células Th2 , Animais , Células Th2/imunologia , Células Th2/efeitos dos fármacos , Antialérgicos/farmacologia , Camundongos , Ácido Ascórbico/farmacologia , Degranulação Celular/efeitos dos fármacos , Células Th1/imunologia , Células Th1/efeitos dos fármacos , Ratos , Hipersensibilidade Alimentar/tratamento farmacológico , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/imunologia , Humanos , Feminino , Masculino , Ovalbumina/imunologia , Ovalbumina/efeitos adversos , Citocinas/metabolismo , Citocinas/imunologia , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Protein adsorption onto nanomaterials often results in denaturation and loss of bioactivity. Controlling the adsorption process to maintain the protein structure and function has potential for a range of applications. Here we report that self-assembled poly(propylene sulfone) (PPSU) nanoparticles support the controlled formation of multicomponent enzyme and antibody coatings and maintain their bioactivity. Simulations indicate that hydrophobic patches on protein surfaces induce a site-specific dipole relaxation of PPSU assemblies to non-covalently anchor the proteins without disrupting the protein hydrogen bonding or structure. As a proof of concept, a nanotherapy employing multiple mast-cell-targeted antibodies for preventing anaphylaxis is demonstrated in a humanized mouse model. PPSU nanoparticles displaying an optimized ratio of co-adsorbed anti-Siglec-6 and anti-FcεRIα antibodies effectively inhibit mast cell activation and degranulation, preventing anaphylaxis. Protein immobilization on PPSU surfaces provides a simple and rapid platform for the development of targeted protein nanomedicines.
Assuntos
Mastócitos , Nanopartículas , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Animais , Camundongos , Adsorção , Humanos , Nanopartículas/química , Nanomedicina/métodos , Anafilaxia , Polipropilenos/química , Degranulação Celular/efeitos dos fármacosRESUMO
Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Ascite , Células Matadoras Naturais , Neoplasias Ovarianas , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ascite/imunologia , Feminino , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Imunoglobulinas/metabolismo , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Degranulação Celular/imunologia , Degranulação Celular/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Cetuximab/farmacologiaRESUMO
Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function. Therefore, in this study, we investigated the effects of diazinon on mast cell function in rat basophilic leukemia (RBL)-2H3 cells. Surprisingly, we found that diazinon inhibited mast cell activation, although the degree of inhibition varied with concentration. Diazinon induced reactive oxygen species (ROS) generation and HO-1 expression at a concentration of 150⯵M without affecting cell viability. Diazinon inhibited A23187-mediated degranulation and Tnf and Il4 expression in RBL-2H3 cells but did not affect calcium influx. Suppression of degranulation by diazinon was reversed when the culture supernatant was removed. As a signaling event downstream of calcium influx, diazinon inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) induced by A23187, whereas the phosphorylation of p38 had little effect. IgE cross-linking-mediated degranulation as well as the induction of Tnf and IL4 expression was significantly inhibited by diazinon, while diazinon had little effect on calcium influx. In conclusion, diazinon inhibited mast cell activation, including degranulation and cytokine expression. When evaluating the in vivo effects of diazinon, its potential to inhibit mast cell activation should be considered in the pathophysiology and development of allergic diseases in terms of basic and clinical aspects, respectively, although the effect of diazinon varies depending on the cell type.
Assuntos
Degranulação Celular , Citocinas , Diazinon , Inseticidas , Mastócitos , Diazinon/toxicidade , Animais , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ratos , Citocinas/metabolismo , Inseticidas/toxicidade , Degranulação Celular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Vitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation. METHODS: Substance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation. RESULTS: VK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of ß-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced ß-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation. CONCLUSION: The anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Mastócitos , Mastócitos/imunologia , Mastócitos/efeitos dos fármacos , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Camundongos , Humanos , Substância P/metabolismo , Degranulação Celular/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Hipersensibilidade/imunologia , Hipersensibilidade/tratamento farmacológico , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Feminino , Linhagem Celular , beta-N-Acetil-Hexosaminidases/metabolismo , Modelos Animais de DoençasRESUMO
Objectives: Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. Methods: HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC ß-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Results: Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. Conclusion: These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.
Assuntos
Degranulação Celular , Cisteína , Galectinas , Mastócitos , Dióxido de Enxofre , Animais , Degranulação Celular/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/imunologia , Mastócitos/efeitos dos fármacos , Cisteína/metabolismo , Ratos , Dióxido de Enxofre/farmacologia , Dióxido de Enxofre/metabolismo , Humanos , Galectinas/metabolismo , Camundongos , Masculino , Anafilaxia Cutânea Passiva , Linhagem CelularRESUMO
The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Streptococcus pyogenes , Estreptolisinas , Humanos , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Inflamação/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/metabolismoRESUMO
This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced ß-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced ß-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.
Assuntos
Antialérgicos , Degranulação Celular , Imunoglobulina E , Extratos Vegetais , Folhas de Planta , Baço , Animais , Extratos Vegetais/farmacologia , Ratos , Baço/efeitos dos fármacos , Baço/imunologia , Baço/citologia , Folhas de Planta/química , Camundongos , Linhagem Celular Tumoral , Degranulação Celular/efeitos dos fármacos , Imunoglobulina E/sangue , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Leucemia Basofílica Aguda/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Imunoglobulina G , Masculino , Interleucina-4/metabolismoRESUMO
BACKGROUND: Phytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6's antipruritic properties. METHODS: The calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the ß-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. RESULTS: Using HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. CONCLUSIONS: Our findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.
Assuntos
Degranulação Celular , Histamina , Mastócitos , Receptores Acoplados a Proteínas G , Canais de Cátion TRPV , Animais , Humanos , Masculino , Camundongos , Antipruriginosos/farmacologia , Antipruriginosos/uso terapêutico , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Células HEK293 , Histamina/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/metabolismo , Prurido/tratamento farmacológico , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Esfingosina/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
The incidence of allergic reactions has risen steadily in recent years, prompting growing interest in the identification of efficacious and safe natural compounds that can prevent or treat allergic diseases. Phellodendron amurense Rupr. has long been applied as a treatment for allergic diseases, whose primary component is phellodendrine. However, the efficacy of phellodendrine as a treatment for allergic diseases remains to be assessed. Mast cells are the primary effectors of allergic reactions, which are not only activated by IgE-dependent pathway, but also by IgE-independent pathways via human MRGPRX2, rat counterpart MRGPRB3. As such, this study explored the effect and mechanism of phellodendrine through this family receptors in treating allergic diseases in vitro and in vivo. These analyses revealed that phellodendrine administration was sufficient to protect against C48/80-induced foot swelling and Evans blue exudation in mice, and suppressed C48/80-induced RBL-2H3 rat basophilic leukemia cells degranulation, and ß-HEX, HIS, IL-4, and TNF-α release. Moreover, phellodendrine could reduce the mRNA expression of MRGPRB3 and responsiveness of MRGPRX2 by altering its structure. It was able to decrease Ca2+ levels, phosphorylation levels of CaMK, PLCß1, PKC, ERK, JNK, p38, and p65, and inhibit the degradation of IκB-α. These analyses indicate that berberine inhibits the activation of PLC and downregulates the release of Ca2+ in the endoplasmic reticulum by altering the conformation of MRGPRB3/MRGPRX2 protein, thereby inhibiting the activation of PKC and subsequently inhibiting downstream MAPK and NF-κB signaling, ultimately suppressing allergic reactions. There may thus be further value in studies focused on developing phellodendrine as a novel anti-allergic drug.
Assuntos
Degranulação Celular , Hipersensibilidade , Mastócitos , Receptores Acoplados a Proteínas G , Animais , Ratos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Degranulação Celular/efeitos dos fármacos , Camundongos , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Citocinas/metabolismo , p-Metoxi-N-metilfenetilamina , Masculino , Phellodendron/química , Linhagem Celular Tumoral , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores de NeuropeptídeosRESUMO
Background: The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). Materials and methods: The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. Results: Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. Conclusions: Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases (AU)
Assuntos
Animais , Masculino , Camundongos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Dissacarídeos/farmacologia , Eicosanoides , Quercetina/análogos & derivados , Mediadores da Inflamação , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fosfolipase C gama/metabolismo , Fosfolipases A2/metabolismo , Fosforilação/efeitos dos fármacos , Quercetina/farmacologiaRESUMO
Abstract Purpose: The effect of a prophylactic oleuropein-rich diet before anesthesia accompanied by the widely-used steroid-based neuromuscular drug rocuronium on mast cell activation was investigated in the study. Methods: 14 rabbits used in the study. The rabbits in the oleuropein group were given oleuropein-rich extract added to the animals' water at doses of 20 mg/kg oleuropein for 15 days orally. After 15 days, all rabbits in the two groups were given general anesthesia with rocuronium of 1 mg/kg. After 1 day, animals were sacrificed and the liver tissue sections stained with H&E, toluidine blue and tryptase for immunohistochemical study. Results: There was no statistically significant difference between ALT, AST and albumin averages of the oleuropein and control groups (p> 0.05). The tryptase average of the control group was higher than the tryptase average of the oleuropein group and this difference was statistically significant (p=0.003). The T. blue average in the oleuropein group was higher than the control group. However, there was no statistically significant difference between groups (p=0.482). Conclusions: Rocuronium adverse effects, like hypersensitivity and anaphylaxis, may limit routine use of this substance. The use of oleuropein reduced the number of inflammatory cells and prevented degranulation.
Assuntos
Animais , Masculino , Coelhos , Fármacos Neuromusculares não Despolarizantes/efeitos adversos , Iridoides/administração & dosagem , Rocurônio/efeitos adversos , Anestesia Geral/efeitos adversos , Mastócitos/efeitos dos fármacos , Anti-Inflamatórios/administração & dosagem , Aspartato Aminotransferases/sangue , Albumina Sérica/análise , Distribuição Aleatória , Degranulação Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão , Dietoterapia/métodos , Alanina Transaminase/sangue , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Profilaxia Pré-Exposição/métodos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Mastócitos/patologiaRESUMO
OBJECTIVE: Formaldehyde exposure during the menstrual cycle is known to affect the course of allergic lung inflammation. Because our previous data demonstrated that formaldehyde combined with an ovariectomy reduced allergic lung inflammation, we investigated the putative role of ovary removal and progesterone treatment when considering the effect of formaldehyde on allergic lung inflammation. METHOD: Ovariectomized rats and their matched controls were exposed to formaldehyde (1%, 3 days, 90 min/day) or vehicle, and immediately after exposure, the rats were sensitized to ovalbumin by a subcutaneous route. After 1 week, the rats received a booster by the same route, and after an additional week, the rats were challenged with ovalbumin (1%) by an aerosol route. The leukocyte numbers, interleukin-10 (IL-10) release, myeloperoxidase activity, vascular permeability, ex vivo tracheal reactivity to methacholine and mast cell degranulation were determined 24 h later. RESULTS: Our results showed that previous exposure to formaldehyde in allergic rats decreased lung cell recruitment, tracheal reactivity, myeloperoxidase activity, vascular permeability and mast cell degranulation while increasing IL-10 levels. Ovariectomy only caused an additional reduction in tracheal reactivity without changing the other parameters studied. Progesterone treatment reversed the effects of formaldehyde exposure on ex vivo tracheal reactivity, cell influx into the lungs and mast cell degranulation. CONCLUSION: In conclusion, our study revealed that formaldehyde and ovariectomy downregulated allergic lung inflammation by IL-10 release and mast cell degranulation. Progesterone treatment increased eosinophil recruitment and mast cell degranulation, which in turn may be responsible for tracheal hyperreactivity and allergic lung inflammation. .