RESUMO
UVB radiation can lead to skin photodamage, which might arise from keratinocyte (KC) activation. Nuclear factor kappa B (NF-κB) assumes an essential function in the context of UVB-triggered skin photodamage. Initiating the NF-κB cascade leads to the release of inflammatory factors from KCs. Livin can modulate both KC activation and function, yet it remains uncertain whether and how Livin regulates KC activation induced by UVB. To explore the involvement of Livin in UVB-triggered skin photodamage and its impact on skin damage through NF-κB activation. Immunofluorescence staining was used to analyse the expression of Livin in individuals with skin photodamage and in mice treated with UVB radiation. KC-specific Livin knockout (LivinΔKC ) mice and HaCaT cells with Livin knockdown were employed to examine the function of Livin in regulating KC activation induced by UVB radiation. Additionally, the impact of Livin on the NF-κB cascade during KC activation was confirmed via western blot analysis. In patients with skin photodamage, UVB-treated mice and HaCaT cells, Livin expression was reduced in KCs. LivinΔKC mice displayed heightened sensitivity to UVB radiation, resulting in more pronounced skin damage and inflammatory responses compared to the control Livinfl/fl mice. Following UVB exposure, both LivinΔKC mice and Livin-knockdown HaCaT cells released elevated levels of cytokines compared to their respective controls. Moreover, the UVB-induced activation of NF-κB in HaCaT cells was significantly enhanced following Livin knockdown. Our findings propose that Livin within KCs could contribute to reducing UVB-induced skin photodamage by regulating the NF-κB pathway.
Assuntos
NF-kappa B , Pele , Animais , Humanos , Camundongos , Queratinócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Pele/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
BACKGROUND: Psoriasis is a prevalent, long-term skin condition characterized by inflammation. Keratinocytes (KCs) are important effector cells that release inflammatory factors and chemokines to promote the inflammatory cascade in psoriasis. However, the mechanisms underlying the activation of KCs in psoriasis remain unclear. Livin suppresses apoptotic proteins and directly affects the growth and spread of cancer cells. Livin expression reportedly increases significantly in lesions of patients with psoriasis; however, its specific role in KC activation remains unknown. This study aimed to examine the impact of Livin on KC activation and the subsequent release of inflammatory mediators. METHODS: Immunofluorescence staining, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and western blotting were used to assess Livin expression in patients with psoriasis, an imiquimod (IMQ)-induced psoriasis-like mouse model, and M5-treated HaCaT cells. To investigate the role of Livin in KCs, we performed RNA sequencing and proteomic analysis of Livin-knockdown (knockdown-HaCaT) and negative control (NC-HaCaT) cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used for enrichment analyses. Moreover, the effect of Livin expression on the release of inflammatory mediators in KCs was verified using ELISA. RESULTS: Livin expression was higher in KCs of patients with psoriasis than in those healthy controls. Livin expression in HaCaT cells treated with M5 increased significantly over time. Livin expression was higher in the skin lesions of the IMQ mouse model than in the control group. Proteomic analysis and RNA sequencing used to investigate the function of Livin in HaCaT cells revealed its potential role in mediating KC activation and inflammatory mediator release, which affected the pathology of psoriasis. CONCLUSIONS: Livin expression played an effect on KCs activation, which induced release of inflammatory mediators and up-regulation of keratin. This study provides a new effector molecule for the mechanism of inflammatory response in psoriasis.
Assuntos
Psoríase , Dermatopatias , Animais , Humanos , Camundongos , Proliferação de Células , Modelos Animais de Doenças , Imiquimode/efeitos adversos , Imiquimode/metabolismo , Mediadores da Inflamação/efeitos adversos , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Proteômica , Psoríase/patologia , Dermatopatias/metabolismoRESUMO
Hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders, but often causes skin irritation in clinic. Mast cell plays an important role in contact dermatitis and triggering pseudo-allergic reactions via MRGPRX2. Whether HQ-induced skin irritant reaction through activating mast cells via MRGPRX2 remains unknown. To investigate the role of mast cells in HQ-induced skin irritant reaction and verify whether MRGPRX2 participated in the HQ effect on mast cells which contributed to the pathogenesis of skin irritant reaction, a mouse model of HQ-induced skin irritation was established to observe the local and systemic inflammation associated with mast cell receptor MrgprB2. Human mast cell LAD2 was used to verify the effect of HQ on mast cells via MRGPRX2 by knocking down with siRNA. As a result, mast cells were involved in the development of HQ-induced irritant reaction, and local inflammation is closely related to mast cell receptor MrgprB2. HQ could activate mast cells via MRGPRX2, causing changes in calcium concentration, degranulation and release of inflammatory cytokines which lead to skin irritant reaction. In conclusion, HQ-induced skin irritant reaction could be skin pseudo-allergic reactions achieved by activating mast cells via MRGPRX2.
Assuntos
Dermatite Atópica , Hipersensibilidade , Animais , Camundongos , Humanos , Mastócitos/patologia , Irritantes/toxicidade , Hidroquinonas/efeitos adversos , Receptores Acoplados a Proteínas G/genética , Inflamação/patologia , Dermatite Atópica/patologia , Degranulação Celular , Proteínas do Tecido Nervoso/genética , Receptores de Neuropeptídeos/genéticaRESUMO
Psoriasis is a chronic inflammatory skin disease. Mast cells are significantly increased and activated in psoriatic lesions and are involved in psoriatic inflammation. Some endogenous substances can interact with the surface receptors of mast cells and initiate the release of downstream cytokines that participate in inflammatory reactions. Neuroblast differentiation-associated protein (AHNAK) is mainly expressed in the skin, esophagus, kidney, and other organs and participates in various biological processes in the human body. AHNAK and its derived peptides have been reported to be involved in the activation of mast cells and other immune processes. This study aimed to investigate whether AHNAK (5758-5775), a neuroblast differentiation-associated protein-derived polypeptide, could be considered a new endogenous substance in psoriasis patients, which activates mast cells and induces the skin inflammatory response contributing to psoriasis. Wild-type mice were treated with AHNAK(5758-5775) to observe the infiltration of inflammatory cells in the skin and cytokine release in vivo. The release of inflammatory mediators by mouse primary mast cells and the laboratory of allergic disease 2 (LAD2) human mast cells was measured in vitro. Molecular docking analysis, molecular dynamics simulation, and siRNA transfection were used to identify the receptor of AHNAK(5758-5775). AHNAK(5758-5775) could cause skin inflammation and cytokine release in wild-type mice and activated mast cells in vitro. Moreover, suppression of tumorigenicity 2 (ST2) might be a key receptor mediating AHNAK(5758-5775)'s effect on mast cells and cytokine release. We propose a novel polypeptide, AHNAK(5758-5775), which induces an inflammatory reaction and participates in the occurrence and development of psoriasis by activating mast cells.
Assuntos
Dermatite , Psoríase , Humanos , Camundongos , Animais , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Mastócitos , Simulação de Acoplamento Molecular , Inflamação/metabolismo , Citocinas/metabolismo , Diferenciação Celular , Proteínas de Membrana/genética , Proteínas de Neoplasias/metabolismoRESUMO
6-Methoxydihydrosanguinarine (6-MDS) is a natural benzophenanthridine alkaloid extracted from Hylomecon japonica (Thunb.) Prantl. It is the first time to explore the effect and mechanism of 6-MDS in breast cancer. Network pharmacology, molecular docking, and molecular dynamics simulation technology were adopted to identify the potential targets and pathways of 6-MDS in breast cancer. Besides, cell proliferation, apoptosis, and western blotting assays were conducted to investigate the effect of 6-MDS on MCF-7 cells. Network pharmacology, molecular docking, and molecular dynamics simulation results confirmed the effect of 6-MDS on resisting breast cancer via the PI3K/AKT/mTOR signaling pathway. In addition, the functional experiments results demonstrated that 6-MDS inhibited proliferation and induced apoptosis and autophagy. The autophagy inhibitor chloroquine and the silence of Atg5 augmented the effect of 6-MDS on promoting apoptosis. Furthermore, 6-MDS suppressed the PI3K/AKT/mTOR signaling pathway, and the PI3K inhibitor LY294002 enhanced these changes and promoted the 6-MDS pro-apoptotic and autophagy effects. 6-MDS triggered the generation of reactive oxygen species. The pretreatment with antioxidant N-acetyl-L-cysteine reversed the changes induced by 6-MDS, including increases in apoptosis and autophagy and inhibition of the PI3K/AKT/mTOR pathway. In conclusion, 6-MDS induces the apoptosis and autophagy of MCF-7 cells by ROS accumulation to suppress the PI3K/AKT/mTOR signaling pathway.
Assuntos
Neoplasias da Mama , Proteínas Proto-Oncogênicas c-akt , Humanos , Feminino , Benzofenantridinas/farmacologia , Benzofenantridinas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células MCF-7 , Neoplasias da Mama/tratamento farmacológico , Simulação de Acoplamento Molecular , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Apoptose , AutofagiaRESUMO
Psoriasis is a chronic, relapsing, immune-mediated, and papulosquamous skin disorder. Excessive mast cell activation, in psoriatic lesions, contributes to inflammation. Various endogenous peptides can participate in the pathogenesis of inflammatory diseases by activating mast cells. Suprabasin (SBSN) is expressed in multiple epithelial tissues and it regulates the normal epidermal barrier function. We have recently shown that suprabasin-derived polypeptides, SBSN(50-63), are significantly increased in psoriatic lesions, through differential peptide analysis. This study was conducted to clarify whether SBSN(50-63) plays a pivotal role in activating mast cells and mediating proinflammatory cytokines and chemokines production in psoriasis. The increased expression of SBSN in psoriatic lesions was confirmed by bioinformatics analysis, PCR and ELISA. Wild-type mice injected subcutaneously with SBSN(50-63) exhibited infiltration of inflammatory cells and the release of cytokines in vivo. SBSN(50-63) stimulated mouse primary mast cells (MPMC) and the laboratory of allergic disease 2 (LAD2) human mast cells to produce more inflammatory mediators than the control group, which were measured ex vivo and in vitro. Toll-like receptor 4 was identified as the receptor of SBSN on mast cells by molecular docking analysis, molecular dynamics simulation, and siRNA transfection. Collectively, SBSN(50-63) could activate mast cells through TLR4, which may increase the inflammatory response in psoriasis.
Assuntos
Mastócitos , Psoríase , Humanos , Animais , Camundongos , Mastócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , Simulação de Acoplamento Molecular , Citocinas/metabolismo , Psoríase/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates mast cells (MCs) activation, which is a key target for the treatment of allergic diseases. However, there are few drugs targeting MRGPRX2. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) is a negative regulator of FcεRΙ-mediated MC activation. However, the regulatory effect of CD300f on MRGPRX2 remains unclear. Dehydroandrographolide (DA) is a main contributor of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) have been shown to inhibit type I hypersensitivity. The aim of this study was to determine whether DA negatively regulated MRGPRX2-mediated MC activation via CD300f and showed therapeutic effect on pseudo-allergic reactions. Mouse allergic models and MC degranulation were detected in vivo and in vitro, and inflammatory mediators were detected. siRNA interference and Biacore were used to verify the target. DA inhibited pseudo-allergic reactions by reducing vasodilation and serum cytokine levels in mice and inhibited MRGPRX2-mediated MC activation. The regulatory effect of DA was significantly decreased after the knockdown of CD300f expression. Moreover, DA upregulated the phosphorylation level of Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2, which are key kinases in the negative regulatory signaling pathways associated with CD300f. In conclusion, DA negatively regulates MRGPRX2-mediated MC activation via CD300f to inhibit pseudo-allergic reactions.
Assuntos
Hipersensibilidade , Animais , Degranulação Celular , Modelos Animais de Doenças , Diterpenos , Hipersensibilidade/tratamento farmacológico , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Luteolin is a flavonoid found in many fruits, vegetables, and herbs. The antiinflammatory effects of luteolin have been reported. In this study, the effect of luteolin on allergic diseases and the underlying molecular mechanism were investigated. We found that luteolin inhibits Fc epsilon RΙ (FcεRΙ)- and Mas-related G protein-coupled receptor X2 (MRGPRX2)-mediated mast cells (MCs) activation, including degranulation and release of cytokines in vitro. Moreover, luteolin reduces the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner. The concentrations of histamine, TNF-α, MCP-1, IL-8, and IL-13 in mice serum are also decreased by luteolin administration. Our study reveals that luteolin can inhibit FcεRΙ- and MRGPRX2-mediated allergic responses in vivo and in vitro, and our results discover luteolin inhibited mast cells mediated anaphylactic reaction by inhibiting the phosphorylation level of PLCγ.
Assuntos
Anafilaxia , Sinalização do Cálcio , Anafilaxia/tratamento farmacológico , Animais , Cálcio/metabolismo , Degranulação Celular , Linhagem Celular , Luteolina/farmacologia , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Intrathecal morphine infusion is often applied to treat chronic pain related to cancer and other conditions. However, persistent pain can be caused by nerve compression because of granuloma formation. In this study, a mouse model of morphine-induced granuloma formation by intrathecal catheterization morphine infusion into the atlanto-occipital membrane of the foramen magnum was established in wild-type mice, MrgprB2 mutant (MrgprB2-/-) mice, and in mast cell-deficient W-sash c-kit mutant (KitW-sh/W-sh) mice. Heat-related pain after surgery was performed to investigate the antipain effect of morphine. H&E staining and immunofluorescence staining of the spinal cord were applied to analyze the mechanism of granuloma formation. Morphine-induced mast cell degranulation was assessed by measuring the Ca2+ influx and mediator release. Anaphylactoid reactions were measured after s.c. morphine infusion to the paws. Chemokine release by mast cells was determined by Human XL Cytokine Array. Experiments with wild-type, MrgprB2 mutant, and mast cell-deficient W-sash c-kit mutant mice demonstrated that morphine activated mast cells and inflammatory cell aggregation through MrgprB2 in intrathecal infusion sites. The chemokine production of human mast cells demonstrated that granuloma formation is correlated with chemokines release. In addition, morphine activated mouse primary mast cells and de novo chemokine synthesis via the MRGPRX2 in human LAD2 cells. We concluded that granuloma formation during intrathecal morphine infusion was associated with MrgprB2/X2. Reducing MRGPRX2 potentially blocks morphine-induced side effects, including granuloma formation.
Assuntos
Granuloma/imunologia , Mastócitos/imunologia , Morfina/efeitos adversos , Dor/imunologia , Receptores Acoplados a Proteínas G/imunologia , Medula Espinal/imunologia , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Forame Magno/imunologia , Forame Magno/patologia , Granuloma/patologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Injeções Espinhais , Masculino , Mastócitos/patologia , Camundongos , Camundongos Knockout , Morfina/farmacologia , Dor/tratamento farmacológico , Dor/patologia , Receptores Acoplados a Proteínas G/genética , Medula Espinal/patologiaRESUMO
Increasing the level of reactive oxygen species (ROS) in cancer cells has been suggested as a viable approach to cancer therapy. Our previous study has demonstrated that mitochondria-targeted flavone-naphthalimide-polyamine conjugate 6c elevates the level of ROS in cancer cells. However, the detailed role of ROS in 6c-treated cancer cells is not clearly stated. The biological effects and in-depth mechanisms of 6c in cancer cells need to be further investigated. In this study, we confirmed that mitochondria are the main source of 6c-induced ROS, as demonstrated by an increase in 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox fluorescence. Compound 6c-induced mitochondrial ROS caused mitochondrial dysfunction and lysosomal destabilization confirmed by absolute quantitation (iTRAQ)-based comparative proteomics. Compound 6c-induced metabolic pathway dysfunction and lysosomal destabilization was attenuated by N-acetyl-L-cysteine (NAC). iTRAQ-based comparative proteomics showed that ROS regulated the expression of 6c-mediated proteins, and treatment with 6c promoted the formation of autophagosomes depending on ROS. Compound 6c-induced DNA damage was characterized by comet assay, p53 phosphorylation, and γH2A.X, which was diminished by pretreatment with NAC. Compound 6c-induced cell death was partially reversed by 3-methyladenine (3-MA), bafilomycin (BAF) A1, and NAC, respectively. Taken together, the data obtained in our study highlighted the involvement of mitochondrial ROS in 6c-induced autophagic cell death, mitochondrial and lysosomal dysfunction, and DNA damage.
Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Naftalimidas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Autofagossomos/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Naftalimidas/química , Proteoma/análise , Proteômica/métodos , Proteína Sequestossoma-1/metabolismoRESUMO
Angioedema may occur during local anesthetic (LA) injection in the perioperative period. Histaminergic angioedema is the most common form of angioedema. It has been reported that LA is a potential exogenous ligand for histamine receptor 1 (H1R). Whether H1R participates in LA-induced angioedema is still controversial. By using a constructed H1R high-expressed cell model, siRNA transfection, pharmacologic means, and genetically modified animal models, here we showed that H1R mediated LA-induced hyperpermeability. LA with uncycled N-methyl scaffold in the side chain (procaine, tetracaine and lidocaine) had a better strength of drug-H1R affinity than that for LA with cycled N atom (bupivacaine and ropivacaine) by the molecular docking assay and equilibrium dissociation constant (KD values) obtained from the cell membrane chromatography (CMC) relative standard method. Procaine, tetracaine, and lidocaine triggered big calcium mobilization in H1R-HEK293 cells and human umbilical vein endothelial cells (HUVECs) but much weaker in NC-HEK293 cells or H1R knockdown HUVECs. Besides, the results of transendothelial resistance measurement, paracellular flux assay and immunofluorescence showed that procaine induced H1R-dependent hyperpermeability, which involved in PLCγ/IP3R/PKC, ERK1/2, Akt signaling pathways, downstream vascular endothelial cadherin (VE-cad) destabilization. Furthermore, H1R gene knockout prevented paw swelling and vascular leakage caused by procaine, tetracaine, and lidocaine in vivo. This study supported a key role of H1R in LA-induced angioedema, and suggested that in the design of LA structure, the ring formation of the N-methyl scaffold on the side chain can properly avoid the angioedema.
Assuntos
Anestésicos Locais/farmacologia , Angioedema/patologia , Permeabilidade Capilar/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Animais , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Mastócitos , Camundongos , Camundongos Knockout , Distribuição AleatóriaRESUMO
Mast cells play an important role in inflammatory and allergic diseases. MAS-related G protein-coupled receptor X2 (MRGPRX2) is a novel G protein-coupled receptor in mast cells that mediates drug-induced anaphylactoid reactions. Piperine has been reported to have anti-inflammatory and anti-allergic pharmacological activities. However, whether the pharmacological effects are regulated by MRGPRX2 has not yet been reported. The purpose of this study was to assess the anti-anaphylactoid effect of Piperine and to explore its potential mechanism. The anti-anaphylactoid effect of Piperine was assessed by an in vivo mouse hindpaw extravasation model. Mast cell intracellular calcium mobilization was measured by a calcium imaging assay. An enzyme immunoassay was used to evaluate the release of pro-inflammatory factors from stimulated mast cells. Activated mast cell related signals were assessed by western blot. A cell membrane chromatography assay was used to determine the binding characteristics of Piperine and MRGPRX2. The results showed that Piperine suppressed mast cell intracellular Ca2+ mobilization, inhibited cytokines and chemokines release, and down-regulated the phosphorylation level of phospholipase Cγ1, protein kinase C, inositol 1,4,5-triphate receptor, P38, protein kinase B, and ERK. Meanwhile, Piperine can bind to MRGPRX2 as a specific antagonist. Hence, Piperine can be served as a novel therapeutic drug candidate for MRGPRX2-mediated anaphylactoid reactions.
Assuntos
Alcaloides/química , Anafilaxia/tratamento farmacológico , Benzodioxóis/química , Mastócitos/metabolismo , Piperidinas/química , Alcamidas Poli-Insaturadas/química , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Masculino , CamundongosRESUMO
The classical mast cells degranulation pathway is mediated by FcεRI aggregation and varies in strength among subjects. Dehydroandrographolide (DA) is one of principal components of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) and considered the main contributors of its therapeutic properties, such as anti-tumor. In this study, inhibition of IgE-mediated anaphylactic reactions and anti-inflammatory potential of DA were investigated. The anti-anaphylactic activity of DA was investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. The release of cytokines was measured using ELISA kits. Human Phospho-Kinase Array kit and western blotting were used to explore the related molecular signaling pathways. DA inhibited IgE-mediated mast cell activation, including degranulation and release of cytokines in vitro. Moreover, DA reduced the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner by inhibiting mast cell degranulation. DA obviously reduced the concentrations of histamine, TNF-α, MCP-1, IL-8, IL-13, and IL-4 in mice serum and inhibited IgE-mediated anaphylactic reactions as a potential P-PLCγ inhibitor. Our study reveals that DA can inhibit allergic responses in vivo and in vitro, and it may be regarded as a novel P-PLCγ inhibitor for preventing mast cell-immediate and delayed allergic diseases.
Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diterpenos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Ovalbumina , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismoRESUMO
BACKGROUND: Retinoic acid (RA)-induced dermatitis is the most frequent side-effect limiting its widespread use. However, the exact mechanisms triggering dermatitis are not fully understood, including the role of skin mast cells. The newly discovered Mas-related G-protein-coupled receptor-X2 (MRGPRX2) in mast cells mediates pseudoallergic drug reactions in several types of dermatitis. A possible contribution of MRGPRX2 to contact dermatitis induced by RA has hitherto not been examined. OBJECTIVES: To investigate whether all-trans-RA (ATRA) activates mast cells via MRGPRX2/MrgprB2 (the mouse orthologue), contributing to the pathogenesis of retinoid-induced dermatitis. METHODS: Wild-type (WT) and MrgprB2-/- mice were treated with topical ATRA to observe local inflammation and mast cell degranulation in vivo by the use of haematoxylin and eosin and immunofluorescence staining. Release of histamine and release of ß-hexosaminidase were measured and calcium influx was detected in Laboratory of Allergic Disease 2 (LAD2) cells with specific knockdown targeting MRGPRX2 by small interfering RNA (siRNA) and in primary cells from MrgprB2-/- mice. RESULTS: As compared with WT mice, MrgprB2-/- mice showed resistance to ATRA-triggered contact dermatitis and local inflammatory reactions in the paws. ATRA activated mast cells via the MrgprB2 pathway in murine cells, and via the MRGPRX2 pathway in human mast cells. CONCLUSIONS: ATRA-induced dermatitis could be achieved by activating mast cells via MRGPRX2/MrgprB2, which may provide a potential therapy target to reduce the side-effect.
Assuntos
Degranulação Celular/efeitos dos fármacos , Dermatite de Contato/etiologia , Mastócitos/fisiologia , Receptores Acoplados a Proteínas G/genética , Tretinoína/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Dermatite , Dermatite de Contato/genética , Técnicas de Silenciamento de Genes , Histamina/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Tretinoína/toxicidade , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Mast cells play an essential role in IgE-FcεR1-mediated allergic diseases. Citrus aurantium is a prolific source of flavonoids with various biological activities, including anti-inflammatory, antioxidant, and anti-tumor efficacies. Neohesperidin is a novel flavonoid isolated from the leaves of C. aurantium. In this study, the anti-allergic and anti-inflammatory potentials of neohesperidin were investigated along with its molecular mechanism. The anti-anaphylactic activity of neohesperidin was evaluated through hind paw extravasation study in mice. Calcium imaging was used to assess intracellular Ca2+ mobilization. The levels of cytokines and chemokines were measured using enzyme immunoassay kits. Western blotting was used to explore the related molecular signaling pathways. Neohesperidin suppressed IgE-induced mast cell activations, including degranulation and secretion of cytokines and eicosanoids through inhibiting phosphorylation of Lyn kinase. Neohesperidin inhibited the release of histamine and other proinflammatory cytokines through a mast cell-dependent passive cutaneous anaphylaxis animal model. Histological studies demonstrated that neohesperidin substantially inhibited IgE-induced cellular infiltration and attenuated mast cell activation in skin tissue. In conclusion, our study revealed that neohesperidin could inhibit allergic responses in vivo and in vitro, and the molecule may be regarded as a novel agent for preventing mast cell-immediate and delayed allergic diseases.
Assuntos
Anafilaxia/tratamento farmacológico , Hesperidina/análogos & derivados , Imunoglobulina E/metabolismo , Mastócitos/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Hesperidina/uso terapêutico , Masculino , Mastócitos/metabolismo , CamundongosRESUMO
Mast cells are unique immune cells that function as sentinels in host defence reactions, including immediate hypersensitivity responses and allergic responses. The mast cell-specific receptor named MAS-related G protein-coupled receptor X2 (MRGPRX2) triggers mast-cell degranulation, a key process in anaphylactoid reactions. It is widely observed that antimicrobials can induce pseudo-allergic reactions (i.e. IgE-independent mechanism) with symptoms ranging from skin inflammation to life-threatening systemic anaphylaxis. However, their direct involvement and the mechanisms underlying anaphylactoid reactions caused by antimicrobials have not been demonstrated. Structurally different antimicrobials were screened by Ca2+ imaging using MRGPRX2 overexpressing HEK293 cells. MRGPRX2 related anaphylactoid reactions induced by these components were investigated by body temperature drop and mast cell degranulation assays. We showed that MRGPRX2 is involved in allergic-like reactions to three types of antimicrobials in a dose-dependent manner. However, mast cells lacking the receptor show reduced degranulation. Furthermore, mice without MAS-related G protein-coupled receptor B2 (the orthologous gene of MRGPRX2) exhibited reduced substance-induced inflammation. Interestingly, ß-lactam and antiviral nucleoside analogues did not induce anaphylactic reactions, which were also observed in vitro. These results should alarm many clinicians that such drugs might induce anaphylactoid reactions and provide guidance on safe dosage of these drugs.
Assuntos
Anafilaxia/induzido quimicamente , Anti-Infecciosos/toxicidade , Degranulação Celular/efeitos dos fármacos , Hipersensibilidade a Drogas/imunologia , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Animais , Anti-Infecciosos/imunologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Mivacurium is a non-depolarizing muscle relaxant and widely used as a short-acting anesthetic. Pseudo-allergic reactions to mivacurium occur when it is administered during perioperative anesthesia. These reactions may present a serious threat to the patient's life, particularly in children. METHODS: MAS-related G protein coupled receptor-related pseudo-allergic reactions that were induced by mivacurium were investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. RESULTS: Mivacurium caused pseudo-allergic reactions in wild-type mice by inducing mast cells to release histamine. However, it did not induce a similar phenomenon in KitW-sh/W-sh mice. Furthermore, MrgprB2-knockout mice displayed no inflammatory response to mivacurium. Mivacurium induced LAD2 cell degranulation in a dose-dependent manner. Mivacurium stimulated intracellular calcium ion (Ca2+) influx in MRGPRX2-HEK293 cells but not in NC-HEK293 cells. However, mivacurium induced the release of only low levels of mediators in LAD2 cells transfected with MRGPRX2-targeted small interfering (si)RNA. Notably, cytokine release was not observed in LAD2 cells even when stimulated with high concentrations of mivacurium. CONCLUSION: Mivacurium activated MRGPRX2 and triggered mast cell degranulation, leading to anaphylactoid reactions. However, mivacurium did not induce the release of other cytokines. Therefore, the targeting of MRGPRX2 can potentially block mivacurium-induced adverse drug effects, particularly pseudo-allergic reactions.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Mastócitos/efeitos dos fármacos , Mivacúrio/efeitos adversos , Mivacúrio/imunologia , Receptores Acoplados a Proteínas G/imunologia , Anafilaxia/imunologia , Animais , Cálcio/imunologia , Degranulação Celular/imunologia , Linhagem Celular , Citocinas/imunologia , Células HEK293 , Histamina/imunologia , Humanos , Masculino , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Adverse drug reactions of Danshen injection mainly manifested as pseudoallergic reactions. In the present study, salvianolic acid A and a pair of geometric isomers (isosalvianolic acid C and salvianolic acid C) were identified as pseudoallergic components in Danshen injection by a high-expression Mas-related G protein coupled receptor X2 cell membrane chromatography coupled online with high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Their pseudoallergic activities were evaluated by in vitro assay, which were consistent with the retention times on the cell membrane chromatography column. Salvianolic acid C, the most outstanding compound, was further found to induce pseudoallergic reaction through Mas-related G protein coupled receptor X2. All the results above indicated that the system developed in this study is an effective method for simultaneously analyzing pseudoallergic components, even those with similar structures and the microcomponents in complex samples (salvianolic acid C in Danshen injection).
Assuntos
Medicamentos de Ervas Chinesas/química , Proteínas do Tecido Nervoso/química , Receptores Acoplados a Proteínas G/química , Receptores de Neuropeptídeos/química , Salvia miltiorrhiza/química , Espectrometria de Massas em Tandem/métodos , Alcenos/efeitos adversos , Alcenos/química , Animais , Ácidos Cafeicos/efeitos adversos , Ácidos Cafeicos/química , Linhagem Celular , Membrana Celular/química , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/efeitos adversos , Humanos , Lactatos/efeitos adversos , Lactatos/química , Masculino , Camundongos , Estrutura Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Polifenóis/efeitos adversos , Polifenóis/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/imunologia , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
BACKGROUND: Harpagoside (HAR) is an active component of Scrophularia ningpoensis (SN), which has anti-inflammatory and anti-immune effects. SN is used widely in China to treat various diseases. Recently, SN has been used as a traditional Chinese medicine injection and used clinically. However, allergic responses to these injections are frequently reported. AIM: We examined whether the main component of SN, HAR, is associated with the allergic reaction to SN. METHODS: This study assessed the effects of HAR in mice and mast cell activation to characterize its anaphylactic effects and underlying mechanisms. Mice hindpaw swelling, serum allergy factor detection, enzyme-linked immunosorbent assays, and degranulation assays were performed to measure allergic mediators both in vivo and in vitro. RESULTS: The present study indicated that HAR induced paw swelling, interleukin-6, inositol triphosphate, tumor necrosis factor-α, and histamine increases in mice. Our in vitro data also showed that HAR induced ß-hexosaminidase, inositol triphosphate, and interleukin-6 release, leading to mast cell degranulation. In contrast, neither C48/80 nor HAR induced local anaphylaxis in STOCK KitW-sh/HNihrJaeBsmJNju mice. CONCLUSIONS: HAR is a potential sensitization compound in SN, and these results provide information for the safe clinical use of SN.
Assuntos
Anafilaxia/induzido quimicamente , Anafilaxia/imunologia , Glicosídeos/toxicidade , Imunoglobulina E/imunologia , Piranos/toxicidade , Anafilaxia/patologia , Animais , Modelos Animais de Doenças , CamundongosRESUMO
BACKGROUND: Baige (BG) is a compound Chinese herbal preparation, constituted of different position extracts (ethanol extracts from Pueraria lobate and SFE-CO2 extracts from Radix Angelicae dahuricae) of P. lobata and A. dahurica to treat the brain injury in patients. AIM: The goal of this study was to identify the neuroprotective properties of BG and its principal component mixture (PCM) and verify whether the material basis for BG is its PCM. METHODS: Middle cerebral artery occlusion (MCAO) was operated on male Sprague-Dawley rat for 2 h, different doses of BG or PCM or vehicle were gavaged after 3 h of MCAO. Rats were sacrificed after 30 days treatment. Blood serum inflammation factors and NGF were detected by ELISA. RESULTS: After 30 days of treatment, both BG and PCM interventions reduced the infarct volume, modified neurological severity score (mNSS) in rats, declined IL-1ß and IL-6 levels in the serum, increased NGF level in the serum and recovered the number of Nissl body in injured brain. CONCLUSIONS: Both BG and PCM exert equivalent levels of recovery effect in MCAO on rats; and PCM is the material foundation of BG. This recovery effect is associated with inflammatory inhibition and NGF production.