RESUMO
Allergic conjunctival disease is an immune-mediated inflammatory disease of the conjunctiva. To develop clinically useful drugs, it is necessary to develop quantitative evaluation methods that reflect the clinical symptoms in experimental animal models. Allergic conjunctivitis model mice were systemically sensitised with ovalbumin (OVA) administered intraperitoneally and locally sensitised with OVA eye drops between day 14-28. Next, conjunctivitis induced by ocular administration of OVA solution to sensitised mice was evaluated based on tear volume. Additionally, we evaluated increase in tear volume induced by direct ocular instillation of histamine, compound 48/80, and carrageenan. An increase in antigen-induced tear volume was observed in the mice model. Additionally, direct instillation of histamine, compound 48/80, and carrageenan increased tear volume. Furthermore, levocabastine inhibited the increase in tear volume in antigen-induced allergic conjunctivitis and histamine- and compound 48/80-induced conjunctivitis models. In contrast, betamethasone suppressed carrageenan-induced tear volume but not histamine- or compound 48/80-induced tear volume. Histamine may be involved in increased tear volume in allergic conjunctivitis. Betamethasone is not directly involved in the action of histamine and is thought to suppress increase in tear volume. Evaluation of tear volume in a conjunctivitis mice model is highly quantitative; therefore, it is possible to evaluate drug efficacy. This is considered a useful index compared with conventional methods.
Assuntos
Carragenina , Conjuntivite Alérgica , Modelos Animais de Doenças , Histamina , Ovalbumina , Lágrimas , Animais , Lágrimas/efeitos dos fármacos , Lágrimas/metabolismo , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/induzido quimicamente , Camundongos , Feminino , p-Metoxi-N-metilfenetilamina/farmacologia , Soluções Oftálmicas , Betametasona/farmacologia , Camundongos Endogâmicos BALB C , MasculinoRESUMO
Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.
Assuntos
Prurido , Receptores de Glicina , Medula Espinal , Animais , Prurido/induzido quimicamente , Prurido/metabolismo , Camundongos , Receptores de Glicina/metabolismo , Masculino , Feminino , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Cloroquina/farmacologia , Camundongos Transgênicos , Pele/inervação , Camundongos Endogâmicos C57BL , p-Metoxi-N-metilfenetilamina/farmacologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologiaRESUMO
Basophils and mast cells are specialized effector cells in allergic reactions. Haliotis discus hannai (abalone), is valuable seafood. Abalone male viscera, which has a brownish color and has not been previously reported to show anti-allergic activities, was extracted with acetone. Six different acetone/hexane fractions (0, 10, 20, 30, 40, and 100%) were obtained using a silica column via ß-hexosaminidase release inhibitory activity-guided selection in phorbol myristate acetate and a calcium ionophore, A23187 (PMACI)-induced human basophils, KU812F cells. The 40% acetone/hexane fraction (A40) exhibited the strongest inhibition of PMACI-induced-ß-hexosaminidase release. This fraction dose-dependently inhibited reactive oxygen species (ROS) production and calcium mobilization without cytotoxicity. Western blot analysis revealed that A40 down-regulated PMACI-induced MAPK (ERK 1/2, p-38, and JNK) phosphorylation, and the NF-κB translocation from the cytosol to membrane. Moreover, A40 inhibited PMACI-induced interleukin (IL)-1ß, IL-6, and IL-8 production. Anti-allergic activities of A40 were confirmed based on inhibitory effects on IL-4 and tumor necrosis factor alpha (TNF-α) production in compound (com) 48/80-induced rat basophilic leukemia (RBL)-2H3 cells. A40 inhibited ß-hexosaminidase release and cytokine production such as IL-4 and TNF-α produced by com 48/80-stimulated RBL-2H3 cells. Furthermore, it's fraction attenuated the IgE/DNP-induced passive cutaneous anaphylaxis (PCA) reaction in the ears of BALB/c mice. Our results suggest that abalone contains the active fraction, A40 is a potent therapeutic and functional material to treat allergic diseases.
Assuntos
Anafilaxia , Antialérgicos , Ratos , Camundongos , Masculino , Humanos , Animais , Anafilaxia/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Basófilos/metabolismo , Hexanos , Imunoglobulina E , Acetona , Interleucina-4/metabolismo , Vísceras/metabolismo , Antialérgicos/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologia , beta-N-Acetil-Hexosaminidases , Citocinas/metabolismoRESUMO
The goal of the present work was to develop novel ß-substituted-α-halomethyl acrylates from a methodology in an aqueous phase and to evaluate their bioactivity as potential inhibitors of mast cell activation. Eleven ß-substituted-α-halomethyl acrylates were synthesized through a modified Horner-Wadsworth-Emmons reaction. Compound 48/80 and the calcium ionophore A23187 stimulated the release of ß-hexosaminidase from mast cells. The effect induced by compound 48/80 was inhibited by compound 5 (320 µM) and compound 9 (160 and 320 µM) without causing cytotoxic effects. The effect induced by A23187 was inhibited by compound 5 (40, 80, 160, and 320 µM) without affecting cell viability. The inhibitory effects exhibited by compounds 5 and 9 were more potent than those of the reference compound sodium cromoglycate at the same concentrations. The biochemical results were consistent with the morphological findings obtained by light and transmission electron microscopy. This study reports, for the first time, that the new synthetic compounds methyl (Z)- 2-bromo-3-(furan-3-yl)acrylate (compound 5) and methyl (E)- 2-bromo-3-(3-bromophenyl)acrylate (compound 9) strongly inhibit mast cell degranulation, without affecting cell viability. The implications of these results are relevant as a basis for developing new anti-inflammatory and mast cell stabilizing drugs.
Assuntos
Degranulação Celular , Mastócitos , Calcimicina/farmacologia , Acrilatos/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Formononetin (FNT) is a plant-derived isoflavone natural product with anti-inflammatory, antioxidant, and anti-allergic properties. We showed previously that FNT inhibits immunoglobulin E (IgE)-dependent mast cell (MC) activation, but the effect of FNT on IgE-independent MC activation is yet unknown. Our aim was to investigate the effects and possible mechanisms of action of FNT on IgE-independent MC activation and pseudoallergic inflammation. We studied the effects of FNT on MC degranulation in vitro with a cell culture model using compound C48/80 to stimulate either mouse bone marrow-derived mast cells (BMMCs) or RBL-2H3 cells. We subsequently measured ß-hexosaminase and histamine release, the expression of inflammatory factors, cell morphological changes, and changes in NF-κB signaling. We also studied the effects of FNT in several in vivo murine models of allergic reaction: C48/80-mediated passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and 2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). The results showed that FNT inhibited IgE-independent degranulation of MCs, evaluated by a decrease in the release of ß-hexosaminase and histamine and a decreased expression of inflammatory factors. Additionally, FNT reduced cytomorphological elongation and F-actin reorganization and attenuated NF-κB p65 phosphorylation and NF-κB-dependent promoter activity. Moreover, the administration of FNT alleviated pseudoallergic responses in vivo in mouse models of C48/80-stimulated PCA and ASA, and DNCB-induced AD. In conclusion, we suggest that FNT may be a novel anti-allergic drug with great potential to alleviate pseudoallergic responses via the inhibition of IgE-independent MC degranulation and NF-κB signaling.
Assuntos
Anafilaxia , Antialérgicos , Isoflavonas , Camundongos , Animais , Mastócitos , p-Metoxi-N-metilfenetilamina/farmacologia , NF-kappa B/metabolismo , Degranulação Celular , Dinitroclorobenzeno/metabolismo , Anafilaxia/tratamento farmacológico , Isoflavonas/metabolismo , Imunoglobulina E/metabolismo , Antialérgicos/uso terapêuticoRESUMO
A balance between stiffness and compliance is essential to normal bladder function, and changes in the mechanical properties of the bladder wall occur in many bladder pathologies. These changes are often associated with the release of basic secretagogues that in turn drive the release of inflammatory mediators from mast cells. Mast cell degranulation by basic secretagogues is thought to occur by activating an orphan receptor, Mas-related G protein-coupled receptor B2 (Mrgprb2). We explored the effects of the putative mast cell degranulator and Mrgprb2 agonist Compound 48/80 on urinary bladder wall mechanical compliance, smooth muscle contractility, and urodynamics, and if these effects were mast cell dependent. In wild-type mice, Mrgprb2 receptor mRNA was expressed in both the urothelium and smooth muscle layers. Intravesical instillation of Compound 48/80 decreased intermicturition interval and void volume, indicative of bladder overactivity. Compound 48/80 also increased bladder compliance while simultaneously increasing the amplitude and leading slope of transient pressure events during ex vivo filling and these effects were inhibited by the Mrgprb2 antagonist QWF. Surprisingly, all effects of Compound 48/80 persisted in mast cell-deficient mice, suggesting these effects were independent of mast cells. These findings suggest that Compound 48/80 degrades extracellular matrix and increases urinary bladder smooth muscle excitability through activation of Mrgprb2 receptors located outside of mast cells. Thus, the pharmacology and physiology of Mrgprb2 in the urinary bladder is of potential interest and importance in terms of treating lower urinary tract dysfunction.
Assuntos
Mastócitos , Bexiga Urinária , Camundongos , Animais , Bexiga Urinária/metabolismo , Mastócitos/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologia , Secretagogos/farmacologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Mast cells play essential role in allergic reactions through the process called mast cell degranulation. Recent studies have found that a basic secretagogue compound 48/80 (C48/80) induces non-IgE-mediated mast cell degranulation via activation of human Mas-related G protein-coupled receptor X2 (MRGPRX2) and mouse MrgprB2. Although previous studies have revealed that caffeic acid (CA) and its derivatives possess anti-allergic effects via IgE-dependent manner, it is largely elusive whether these compounds have impact on MRGPRX2/MrgprB2 to exert inhibitory effects. Therefore, the present study investigated whether CA as well as its derivatives - rosmarinic acid (RA) and caffeic acid phenethyl ester (CAPE) - has the ability to inhibit the activity of MRGPRX2/MrgprB2 to evoke pseudo-allergic effects. As a result, it was found that CAPE inhibits C48/80-induced activation of MRGPRX2/MrgprB2, but neither CA nor RA showed discernible inhibition. Furthermore, the ß-hexosaminidase release assay showed that CAPE inhibits mouse peritoneal mast cell degranulation in both IgE-dependent and MrgprB2-dependent manners. Additionally, mouse paw edema induced by C48/80 was dramatically suppressed by co-treatment of CAPE, suggesting that CAPE possesses a protective effect on C48/80-evoked pseudo-allergic reactions. The pretreatment of CAPE also significantly decreased scratching bouts of mice evoked by C48/80, demonstrating that CAPE also has an anti-pruritic effect. Therefore, these data implicate that CAPE can suppress pseudo-allergic reactions evoked by C48/80 via MrgprB2-dependent manner. Finally, molecular docking analysis showed that CAPE is predicted to bind to human MRGPRX2 in the region where C48/80 also binds, implying that CAPE can be a competitive inhibitor of MRGPRX2. In conclusion, it is found that CAPE has the ability to inhibit MRGPRX2/MrgprB2, leading to the prevention of mast cell degranulation and further to the alleviation of mast cell reactions. These results indicate that CAPE as a CA derivative could be developed as a new protective agent that exerts dual inhibition of mast cell degranulation mediated by IgE and MRGPRX2/MrgprB2.
Assuntos
Antialérgicos , Hipersensibilidade , Animais , Antialérgicos/farmacologia , Ácidos Cafeicos , Degranulação Celular , Humanos , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Álcool Feniletílico/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Secretagogos/metabolismo , Secretagogos/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , beta-N-Acetil-Hexosaminidases/farmacologia , p-Metoxi-N-metilfenetilamina/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The activation and degranulation of immune cells play a pivotal role in allergic inflammation, a pathological condition that includes anaphylaxis, pruritus, and allergic march-related diseases. In this study, trifuhalol A, a phlorotannin isolated from Agarum cribrosum, inhibited the degranulation of immune cells and the biosynthesis of IL-33 and IgE in differentiated B cells and keratinocytes, respectively. Additionally, trifuhalol A suppressed the IL-33 and IgE-mediated activation of RBL-2H3 cells through the regulation of the TAK1 and MK2 pathways. Hence, the effect of trifuhalol A on allergic inflammation was evaluated using a Compound 48/80-induced systemic anaphylaxis mouse model and a house dust mite (HDM)-induced atopic dermatitis (AD) mouse model. Trifuhalol A alleviated anaphylactic death and pruritus, which appeared as an early-phase reaction to allergic inflammation in the Compound 48/80-induced systemic anaphylaxis model. In addition, trifuhalol A improved symptoms such as itching, edema, erythema, and hyperkeratinization in HDM-induced AD mice as a late-phase reaction. Moreover, the expression of IL-33 and thymic stromal lymphopoietin, inflammatory cytokines secreted from activated keratinocytes, was significantly reduced by trifuhalol A administration, resulting in the reduced infiltration of immune cells into the skin and a reduction in the blood levels of IgE and IL-4. In summarizing the above results, these results confirm that trifuhalol A is a potential therapeutic candidate for the regulation of allergic inflammation.
Assuntos
Anafilaxia , Dermatite Atópica , Anafilaxia/tratamento farmacológico , Animais , Citocinas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Imunoglobulina E , Inflamação/patologia , Interleucina-33/metabolismo , Mastócitos/metabolismo , Camundongos , Prurido/metabolismo , Pyroglyphidae , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
BACKGROUND Interstitial cystitis (IC) is a recurrent and chronic inflammatory disease that compromises patients' quality of life. Effective treatments for IC are limited. This study aimed to evaluate the therapeutic potency of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in an IC-induced rat model and investigate the potential molecular mechanism in a mast cell model (rat basophilic leukemia cells, RBL-2H3) in treating IC in a coculture system. MATERIAL AND METHODS The rat model of IC was induced by cyclophosphamide (CYP). Rats were randomly divided into 3 groups: sham, IC+PBS, and IC+MSC. In the coculture system, RBL-2H3 cells were sensitized overnight to Compound 48/80 (C48/80), cocultured with UC-MSCs for 3 days, and collected for subsequent experiments. RBL-2H3 cells were randomly divided into 3 groups: sham, C48, and UC-MSCs (C48+MSC). RESULTS The UC-MSCs marked by thymidine analog 5-ethynyl-2-deoxyuridine (EdU) were transplanted in the treatment group, and were densely distributed in the bladder. Accordingly, the conscious cystometry was measured and the bladder tissues were harvested. Compared with the sham group, the treated IC rats exhibited shorter bladder voiding intervals (307±35 vs 217±37 s; P<0.01), more integral epithelia, and less collagen fiber aggregation, infiltration and degranulation of mast cells, and inflammatory cytokines in the bladder tissue. In the coculture system, compared with the C48 group, the UC-MSC-treated RBL-2H3 cells had suppressed degranulation. CONCLUSIONS UC-MSCs treatment showed a promising therapeutic effect on treating IC in vivo and in vitro. UC-MSCs inhibit mast cell degranulation in IC and could be a potential therapeutic target to ameliorate inflammation in IC.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Cistite Intersticial , Mastócitos/imunologia , Cordão Umbilical/citologia , Bexiga Urinária , p-Metoxi-N-metilfenetilamina/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Técnicas de Cocultura/métodos , Cistite Intersticial/imunologia , Cistite Intersticial/terapia , Citocinas/análise , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologia , Micção/imunologiaRESUMO
The aim of this study was to determine whether the lactones dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one, would be effective in an animal model of gastric ulcer induced by mast cell activation. Rats were divided into ten groups. Treatments were repeated for four days. The degree of gastric erosion was assessed with a scoring system and histological preparations. Gastric mast cell morphology was analyzed by histological procedures. Serum serotonin levels were determined as markers of mast cell activation. Statistical analyses were done using ANOVA and Tukey-Kramer test. We demonstrated that the repeated administration of compound 48/80 results in extensive mucosal lesions in the gastric mucosa and that such lesions occurred in association with mast cell degranulation and a significant increase of serum serotonin. We showed that these lesions were prevented by dehydroleucodine, xanthatin, and 3-benzyloxymethyl-5H-furan-2-one and that this effect was similar to that obtained with sodium cromoglycate. In conclusion, the results of the present study indicate that the optimal gastric cytoprotective dose of dehydroleucodine, xanthatin, and 3-benzyloxymethyl-5H-furan-2-one is efficacious in an animal model of gastric ulcer induced by mast cell activation. Our findings suggest that these lactones could be valuable tools for designing novel therapeutic agents for digestive disorders associated with inappropriate mast cell activation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Mastocitose/tratamento farmacológico , Úlcera Gástrica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Furanos/farmacologia , Mucosa Gástrica/patologia , Humanos , Lactonas/farmacologia , Mastocitose/metabolismo , Mastocitose/patologia , Ratos , Sesquiterpenos/farmacologia , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The degranulation of cardiac mast cells is associated with occurrence and development of myocardial ischemia/reperfusion (I/R) injury. Dexmedetomidine has a cardioprotective effect from I/R injury. The purpose of this study was to investigate whether dexmedetomidine preconditioning induced cardioprotection is related to suppression of degranulation of cardiac mast cell. Both in vivo and in vitro experimental results revealed that hemodynamic disorder, arrhythmia, infarct size, histopathological score, and mast cell degranulation were dramatically increased in I/R injury groups compared with non-I/R groups, and mastocyte secretagogue compound 48/80 aggravated these damages, but it can be improved by dexmedetomidine preconditioning. Similarly, compound 48/80 increased levels of cardiac troponin I (cTnI) and tryptase, cardiomyocytes apoptosis, and expression of high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and nuclear factor-kappa B p65 (NF-κB p65) in cardiac tissues induced by I/R injury, but it can be partially decreased by dexmedetomidine pretreatment. Compound 48/80 inhibited proliferation of H9C2(2-1) and RBL-2H3, exacerbated apoptosis of H9C2(2-1), and elevated levels of cTnI and tryptase, while both of which were abolished by dexmedetomidine pretreatment. Our data suggest that dexmedetomidine preconditioning alleviates the degranulation of mast cells and the apoptosis of cardiomyocytes caused by I/R injury, and inhibits the activation of inflammatory related factors HMGB1, TLR4, and NF-κB p65.
Assuntos
Cardiotônicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Dexmedetomidina/farmacologia , Mastócitos/efeitos dos fármacos , Isquemia Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Arritmias Cardíacas/prevenção & controle , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Precondicionamento Isquêmico , Masculino , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/psicologia , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and ß-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.
Assuntos
Degranulação Celular , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Ligantes , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Tetraspanina 30/metabolismo , Tiazolidinas/farmacologia , beta-N-Acetil-Hexosaminidases/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.
Assuntos
Cloroquina/toxicidade , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células do Corno Posterior/fisiologia , Prurido/fisiopatologia , Receptores Adrenérgicos alfa 1/fisiologia , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Masculino , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Prurido/induzido quimicamente , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Adrenérgicos alfa 1/biossíntese , Receptores Adrenérgicos alfa 1/deficiência , Receptores Adrenérgicos alfa 1/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/biossíntese , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Mast cells (MCs) exist intracranially and have been reported to affect higher brain functions in rodents. However, the role of MCs in the regulation of emotionality and social behavior is unclear. In the present study, using male mice, we examined the relationship between MCs and social behavior and investigated the underlying mechanisms. Wild-type male mice intraventricularly injected with a degranulator of MCs exhibited a marked increase in a three-chamber sociability test. In addition, removal of MCs in Mast cell-specific Toxin Receptor-mediated Conditional cell Knock out (Mas-TRECK) male mice showed reduced social preference levels in a three-chamber sociability test without other behavioral changes, such as anxiety-like and depression-like behavior. Mas-TRECK male mice also had reduced serotonin content and serotonin receptor expression and increased oxytocin receptor expression in the brain. These results suggested that MCs may contribute to the regulation of social behavior in male mice. This effect may be partially mediated by serotonin derived from MCs in the brain.
Assuntos
Comportamento Animal/fisiologia , Encéfalo , Mastócitos/fisiologia , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Comportamento Social , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.
Assuntos
Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Pseudomonas/efeitos dos fármacos , Triclosan/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Água Doce/microbiologia , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Oklahoma , Pseudomonas/genética , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S , Microbiologia da Água , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
BACKGROUND: Tripterine (TRI), an active monomer in Tripterygium wilfordii, has significant pharmacological activities, such as anti-inflammatory, immunosuppressive and anti-tumor activities. TRI may be used to treat allergic diseases because of its characteristics of immunosuppression. OBJECTIVE: This study aims to explore the anti-allergic effect of TRI. METHODS: It was tested in vivo and in vitro in this study. RESULTS: The results showed that TRI could significantly inhibit histamine release from rat peritoneal mast cells; the inhibitory effect of TRI on histamine release was stronger than that of other known histamine inhibitors such as disodium cromoglyceride. TRI also significantly inhibited systemic anaphylactic shock induced by compound 48/80 and skin allergy induced by IgE, and inhibited the expression of inflammatory factors secreted by Human Mast Cells (HMC-1) induced by Phorbol 12-Myristate 13- Acetate (PMA) and calcium carrier A23187. In the animal model of allergic rhinitis induced by Ovalbumin (OA), the scores of friction, histamine, IgE, inflammatory factors and inflammatory cells decreased after TRI was administered orally or nasally. CONCLUSION: TRI, as an active immunoregulatory factor, has great potential in the treatment of mast cell-mediated allergic diseases.
Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Rinite Alérgica/tratamento farmacológico , Triterpenos/farmacologia , Animais , Antialérgicos/uso terapêutico , Calcimicina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Masculino , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Triterpenos Pentacíclicos , Ratos , Rinite Alérgica/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Triterpenos/uso terapêutico , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture. METHODS: Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (KitW-sh/W-sh mice). RESULTS: Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus KitW-sh/W-sh mice). CONCLUSIONS: These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.
Assuntos
Aneurisma Roto/imunologia , Aneurisma Intracraniano/imunologia , Mastócitos/imunologia , Aneurisma Roto/patologia , Aneurisma Roto/prevenção & controle , Animais , Catepsina G/genética , Quimases/genética , Cromolina Sódica/farmacologia , Modelos Animais de Doenças , Interleucina-6/genética , Aneurisma Intracraniano/patologia , Masculino , Estabilizadores de Mastócitos/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Hemorragia Subaracnóidea/imunologia , Hemorragia Subaracnóidea/patologia , Hemorragia Subaracnóidea/prevenção & controle , Triptases/genética , Fator de Necrose Tumoral alfa/genética , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
In the present study the effects and molecular mechanisms of wheat bran (WB), the hard outer layer of the wheat kernel used in food ingredients, on mast cell-mediated allergic responses in vitro and in vivo were investigated. The water extract of WB inhibited degranulation and expression of allergic and inflammatory mediators such as tumor necrosis factor-α, cyclooxygenase-2 and inducible nitric oxide synthase in antigen-stimulated RBL-2H3 cells. These anti-allergic activities of WB were mediated by the inactivation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which play important roles in degranulation and expression of various allergic and inflammatory molecules. In agreement with its in vitro effects, WB inhibited immunoglobulin E (IgE)/antigen-induced and compound 48/80-induced anaphylactic reactions in vivo. Taken together, these findings suggest the pharmacological potential of WB in the regulation of allergic diseases, including allergic rhinitis, atopic dermatitis, asthma and anaphylaxis.
Assuntos
Fibras na Dieta/farmacologia , Hipersensibilidade/patologia , Mastócitos/patologia , Extratos Vegetais/farmacologia , Animais , Antígenos/imunologia , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Imunoglobulina E/metabolismo , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Mast cells (MCs) degranulation is a key process during the allergic inflammatory response. MCs release their preformed and new synthesized granules after activation. We found that granules were released partially and selectively after the activation of bone marrow-derived mouse mast cells (BMMCs). Next, we investigated the response of degranulated MCs to a new challenge. BMMCs were activated by antibody/antigen (IgE/Ag) or compound 48/80 (C48/80). The degranulated BMMCs were then reactivated by either IgE/Ag or C48/80 without time intervals. Flow cytometry was used to detect the expression of CD117, FcεRI, and intracellular granules of BMMCs, and BMMCs degranulation was detected using the ß-hexosaminidase release assay. The morphology of BMMCs was observed by staining with toluidine blue. Degranulated BMMCs activated by IgE/Ag failed to respond to the same IgE/Ag challenge and released ß-hexosaminidase independent of unoccupied FcεRI, but responded to C48/80. Degranulated BMMCs activated by C48/80 responded to either IgE/Ag or C48/80. These results indicated that degranulated BMMCs could be reactivated and released granule mediators again, this revealed the unique mediator releasing mechanism of degranulated MCs and their potential function in maintaining inflammation or causing hypersensitivity.
Assuntos
Células da Medula Óssea/imunologia , Degranulação Celular/imunologia , Mastócitos/imunologia , Animais , Antígenos/imunologia , Células da Medula Óssea/citologia , Células Cultivadas , Imunoglobulina E/imunologia , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
OBJECTIVE: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3. METHODS: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry. RESULTS: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 µM) and CP96344 (1-100 µM) suppressed SP (10 µM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 µM)- and compound-48/80 (10 µg/mL)-induced RPMC activation. CONCLUSIONS: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.