Extract of Boehmeria nivea Suppresses Mast Cell-Mediated Allergic Inflammation by Inhibiting Mitogen-Activated Protein Kinase and Nuclear Factor-κB.

Mast cells are effector cells that initiate allergic inflammatory immune responses by inducing inflammatory mediators. Boehmeria nivea (Linn.) Gaudich is a natural herb in the nettle family Urticaceae that possesses numerous pharmacological properties. Despite the various pharmacological benefits of Boehmeria nivea, its effects on allergic inflammation have not yet been determined. Here, we investigated the effect of the ethanol extract of Boehmeria nivea (BNE) on degranulation rat basophilic leukemia (RBL)-2H3 mast cells stimulated with anti-dinitrophenyl (anti-DNP) and bovine serum albumin (BSA) during immunoglobulin E (IgE)-mediated allergic immune response. The results showed inhibition of the release of ß-hexosaminidase and histamine from the cells. BNE suppressed pro-inflammatory cytokines (Tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, and IL-6) and reduced T helper (Th)2 cytokine IL-4 expression and/or secretion correlated with the downregulation of p38, extracellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) signaling pathways in treated RBL-2H3 mast cells. In passive cutaneous anaphylaxis, treatment with BNE during IgE-mediated local allergic reaction triggered a reduction in mouse ear pigmentation and thickness. Taken together, these results indicated that BNE suppressed mast cell-mediated inflammation, suggesting that BNE might be a candidate for the treatment of various allergic disorders.

Cell membrane chromatography coupled online with LC-MS to screen anti-anaphylactoid components from Magnolia biondii Pamp. targeting on Mas-related G protein-coupled receptor X2.

Mas-related G protein-coupled receptor X2 was a mast cell-specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell-mediated anaphylactoid and inflammatory diseases. The anti-anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas-related G protein-coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti-anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti-anaphylactoid components. Bioactivity of these two components were investigated by ß hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit ß hexosaminidase and histamine release in a concentration-dependent manner. This Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti-anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs.

Liquid Chromatography Tandem Mass Spectrometry Based Label-Free Quantification Method for Assessment of Allergen-Induced Anaphylactoid Reactions.

Mast cells are essential in mediating inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and ß-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, and P14R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R2 > 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, and the results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics and to be a major tool of allergic drug assessment or antiallergic drug development in research.

In Vivo Evaluation of Taste-Masked Fast-Disintegrating Sublingual Tablets of Epinephrine Microcrystals.

In community settings, IM injection of 0.3 mg epinephrine (Epi) using an auto-injector is the drug of choice for treatment of anaphylaxis. Previously, a taste-masking (TM) formulation of fast-disintegrating sublingual tablets (FDSTs) was developed in our lab. Also, Epi was micronized (Epi-MC) successfully and reduced the previously achieved bioequivalent sublingual Epi dose to 0.3 mg IM injection by half using non-taste-masked fast-disintegrating sublingual tablets (TM-FDSTs). Our objective for this study was to evaluate the sublingual absorption of Epi-MC using TM-FDST. These sublingual Epi tablets have potential for out-of-hospital treatment of anaphylaxis and are suitable for human studies. TM-FDSTs containing Epi-MC were manufactured by direct compression. The rate and extent of Epi absorption from our developed 20 mg Epi-MC-TM-FDSTs (n = 5) were evaluated in rabbits and compared to the previous result from 20 mg Epi-MC in non-TM-FDSTs and EpiPen® auto-injector. Blood samples were collected over 1 h, and Epi concentrations were measured using HPLC with electrochemical detection. Mean ± SEM AUC0-1 h and Cmax from 20 mg Epi-MC-TM-FDSTs (733 ± 78 ng/ml/min and 30 ± 8 ng/ml) and 20 mg Epi-MC-non-TM-FDSTs (942 ± 109 ng/ml/min and 38 ± 4 ng/ml) were not significantly different (p > 0.05) from each other or from EpiPen® (592 ± 50 ng/ml/min and 28 ± 3 ng/ml) but were significantly higher (p < 0.05) than endogenous Epi after placebo FDSTs (220 ± 32 ng/ml/min and 8 ± 1 ng/ml). Mean ± SD Tmax was not significantly different (p > 0.05) among all formulations. Epi-MC-TM-FDSTs formulation improved Epi absorption twofold and reduced the required bioequivalent dose by 50%, similar to results obtained using non-TM-FDSTs. The incorporation of TM excipients did not interfere with the absorption of Epi-MC.

Deciphering biochemical basis of Qingkailing injection-induced anaphylaxis in a rat model by time-dependent metabolomic profiling based on metabolite polarity-oriented analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingkailing injection (QKLI) is prepared from eight traditional Chinese medicinal materials or their extracts, which is widely used in clinical practice to treat the upper respiratory inflammation, pneumonia, high fever and viral encephalitis, nonetheless, suffering from serious anaphylaxis. AIM OF STUDY: This study aims to develop an integrative metabolomics approach for deciphering the biochemical basis of QKLI induced anaphylaxis (QKLI-IA). MATERIALS AND METHODS: The accuracy of animal modeling, the coverage of detected metabolites and the timeliness of pathological reaction are three key factors for revealing the biochemical basis of disease with untargeted metabolomics. In this study, firstly, the allergic rats (responders) were first screened by passive cutaneous anaphylaxis experiment and then were utilized for modeling. To cover a wider range of metabolites, a large-scale untargeted metabolomics based on metabolites polarity-oriented analysis was performed using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Then, to evaluate the timeliness of QKLI-IA, a time-dependent metabolomic profiling including the early, mid and late anaphylaxis stages of QKLI-IA, was performed. RESULTS: Corresponding to early, mid and late anaphylaxis stages of QKLI-IA, 14, 9 and 4 potential biomarkers were identified, respectively. Metabolism pathway analysis revealed that QKLI-IA resulted in dynamic changes in serum amino acid, fatty acid, glycerolipid, and phospholipid metabolisms. Twenty-four metabolites were found with identical fluctuating trends across the three stages of QKLI-IA. The results indicate that the pathogenesis of QKLI-IA is closely related to arachidonic acid metabolism. CONCLUSION: This research provides a methodology reference for revealing the biochemical basis of disease using metabolomic profiling and offers a new insight to understand the pathogenesis of QKLI-IA.

Eupatilin suppresses the allergic inflammatory response in vitro and in vivo.

INTRODUCTION: Eupatilin, a pharmacologically active ingredient found in Artemisia asiatica, has been reported to have anti-oxidative, anti-inflammatory, and anti-apoptotic activities. However, molecular mechanisms underlying its anti-allergic properties are not yet clear. In this study, we investigated the effects of eupatilin on allergic inflammation in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cells and a compound 48/80-induced anaphylactic shock model. METHODS: Cytokine assays, histamine assays, quantitative real-time polymerase chain reaction analysis, western blot analysis and compound 48/80-induced anaphylactic shock model were used in this study. RESULTS: Eupatilin significantly suppresses the expression and production of pro-inflammatory cytokines, such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 in vitro and in vivo. In addition, eupatilin inhibits nuclear factor kappa B (NF-κB) activation by regulating the phosphorylation and degradation of IκBα via the Akt/IKK(α/ß) pathway. Eupatilin treatment also attenuates the phosphorylation of p38, ERK, and JNK MAPKs. Furthermore, eupatilin blocked anaphylactic shock and decreased the release of histamine. CONCLUSIONS: Anti-allergic inflammation may involve the expression and production of regulating pro-inflammatory cytokines via Akt/IKK(α/ß) and MAPK activation of NF-κB. On the basis of these data, eupatilin is a potential candidate for the treatment of allergic diseases.

Diospyros kaki calyx inhibits immediate-type hypersensitivity via the reduction of mast cell activation.

CONTEXT: Diospyros kaki L. (Ebenaceae) fruit is widely distributed in Asia and is known to exert anti-inflammatory and antithrombotic effects. OBJECTIVE: We evaluated the inhibitory effect of aqueous extract of D. kaki calyx (AEDKC) on mast cell-mediated immediate-type hypersensitivity and underlying mechanism of action. MATERIALS AND METHODS: For in vivo, ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA, AEDKC (1-100 mg/kg) was orally administered 3 times during 14 days. In the PCA, AEDKC was orally treated 1 h before the antigen challenge. The control drug dexamethasone was used to compare the effectiveness of AEDKC. For in vitro, IgE-stimulated RBL-2H3 cells and primary cultured peritoneal mast cells were used to determine the role of AEDKC (0.01-1 mg/mL). RESULTS: Oral administration of AEDKC dose dependently suppressed rectal temperature decrease and increases in serum histamine, total IgE, OVA-specific IgE, and interleukin (IL)-4 in the ASA. In the PCA, AEDKC reduced Evans blue pigmentation. Compared to dexamethasone (10 mg/kg), AEDKC (100 mg/kg) showed similar inhibitory effects in vivo. AEDKC concentration dependently suppressed the release of histamine and ß-hexosaminidase through the reduction of intracellular calcium in mast cells. In addition, AEDKC decreased the expression and secretion of tumour necrosis factor-α and IL-4 by the reduction of nuclear factor-κB. The inhibitory potential of AEDKC (1 mg/mL) was similar with dexamethasone (10 µM) in vitro. CONCLUSIONS: We suggest that AEDKC may be a potential candidate for the treatment of mast cell-mediated allergic diseases.

The Herbal Medicine KIOM-MA128 Inhibits the Antigen/IgE-Mediated Allergic Response in Vitro and in Vivo.

KIOM-MA128, a novel herbal medicine, has been reported to exert some beneficial effects on various biological events, such as atopic dermatitis, inflammation and cancer. The aim of this study is to investigate how KIOM-MA128 regulates the allergic response. We measured the activity of ß-hexosaminidase and the levels of allergic mediators in the conditioned media of antigen/IgE (Ag/IgE)-activated RBL-2H3 mast cells. We examined the levels of proteins associated with both the FcεRI and arachidonate cascades. Finally, we established the passive cutaneous anaphylaxis (PCA) model in mice to confirm the anti-allergic effects of KIOM-MA128 in vivo. KIOM-MA128 dose-dependently inhibited degranulation and the production of the allergic mediators described above, with no significant cytotoxicity. In the arachidonate cascade, KIOM-MA128 significantly reduced both cytosolic phospholipase A2 (cPLA2) phosphorylation and cyclooxygenase-2 (COX-2) expression. Moreover, in the FcεRI cascade, KIOM-MA128 not only inhibited activation of LYN, FYN and SYK, known as the rate-limiting proteins of the FcεRI cascade, but also suppressed the phosphorylation of ERK, p38 and JNK, which is related to cytokine expression. Finally, 50 to 100 mg/kg KIOM-MA128 significantly attenuated the Ag/IgE-induced PCA reaction in mice. These findings provide novel information and improve our understanding of the anti-allergic effects of KIOM-MA128 on allergic diseases.

The Sound of a Buk (Korean Traditional Drum) Attenuates Anaphylactic Reactions by the Activation of Estrogen Receptor-ß.

BACKGROUND: Anaphylaxis is associated with systemic vasodilation that causes low blood pressure and induces hypoxic brain damage. The sound of a Buk (Korean traditional drum) is similar to the human heart beat and affects blood pressure, heart rate, and the nervous system by increasing physiological excitation and sympathetic nervous system activity. So, this study focused on the effect of Buk music as a means of treating anaphylaxis. METHODS: Mice were given an intraperitoneal injection of compound 48/80 (6.5 mg/kg, a mast cell degranulator). After compound 48/80 injection, mice were exposed to Buk music and white noise for 5 min in a sound isolation booth. The mortality rate was checked over the next 40 min. Levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the serum and brain tissues were analyzed by Western blotting, quantitative real-time PCR, and ELISA methods. RESULTS: Exposure to Buk music significantly reduced compound 48/80-induced mortality and histamine release, as well as HIF-1α and VEGF levels compared with the compound 48/80 group or compound 48/80 and white noise group. Buk music also reduced levels of tumor necrosis factor-α, and significantly increased estrogen receptor-ß mRNA levels. CONCLUSION: These results indicate that Buk music has potential for the treatment of anaphylaxis.

Integrative analysis of proteomics and metabolomics of anaphylactoid reaction induced by Xuesaitong injection.

Injection with natural compounds is an important method in the application of natural medicine, but its adverse drug reactions (ADRs) occur frequently, particularly the anaphylactoid reaction, which accounts for more than 77% of all reactions and has become a serious threat to public health. Here, the Xuesaitong injection (XSTI) was employed as an example to elucidate its anaphylactoid mechanism and look for potential biomarkers to assay the anaphylactoid reaction of herbal medicine injection by proteomics and metabolomics. These results disclosed that 13 differential proteins and 28 metabolites, which were further approved using the ELISA method and reference standards, respectively, were suggested as potential biomarkers to examine the anaphylactoid mechanism. The up-regulated expression of Gpx1, Sc5b9, C4d and down-regulated expression of F12, Kng1, C2 and C6 revealed that the XSTI-induced anaphylactoid reaction occurs via direct stimulation, complement and the kallikrein-kinin pathway. In addition, substances that induce an anaphylactoid effect include histamine, LTB4, uric acid and other drugs, which have been confirmed to be involved in arginine and proline metabolism, histidine metabolism, arachidonic acid metabolism purine metabolism and the TCA cycle. Furthermore, separation experiments have indicated that 10-kDa molecules of XSTI are the main allergenic factor inducing an anaphylactoid reaction.

Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy.

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA)-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g.) exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs), and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-κB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-κB in BMMCs. In summary, our data demonstrates a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals.

The antiallergic mechanisms of Citrus sunki and bamboo salt (K-ALL) in an allergic rhinitis model.

The antiallergic effects of traditional medicines have long been studied. Traditional Korean medicine, Citrus sunki and bamboo salt, has been used for the treatment of allergic diseases in Korea. K-ALL, composed of Citrus sunki and bamboo salt, is a newly prepared prescription for allergic patients. To develop the new antiallergic agent, we examined the effects of K-ALL through in vivo and in vitro models. K-ALL and naringin (an active compound of K-ALL) significantly inhibited histamine release from rat peritoneal mast cells. This inhibitory effect of K-ALL on histamine release was higher than effects from other known histamine inhibitors such as bamboo salt, Citrus sunki or disodium cromoglycate. K-ALL significantly inhibited systemic anaphylactic shock induced by the compound 48/80 and passive cutaneous anaphylaxis induced by the IgE. K-ALL also inhibited production and mRNA expression of inflammatory cytokines induced by phorbol 12-myristate 13-acetate and the calcium ionophore A23187 on HMC-1 cells (a human mast cell line). In the ovalbumin-induced allergic rhinitis animal model, rub scores, histamine, IgE, inflammatory cytokines and inflammatory cell counts were all reduced by the oral or nasal administration of K-ALL (pre and posttreatment). These results indicate the great potential of K-ALL as an active immune modulator for the treatment of mast cell-mediated allergic diseases.

Supplementary seleno-L-methionine suppresses active cutaneous anaphylaxis reaction.

To clarify the relationship between selenium supplementation and type I allergic reaction, we investigated the effect of seleno-L-methionine (SeMet) supplementation on the active cutaneous anaphylaxis (ACA) reaction and cytokine production in splenocytes. Female BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), and SeMet was administered orally for 2 weeks followed by a challenge with OVA to induce an ACA reaction. SeMet supplementation suppressed the ACA reaction in a dose-dependent manner. Plasma OVA-specific immunoglobulin E (IgE) level was strongly inhibited in SeMet-supplemented mice compared with control mice. The mRNA expression levels of the T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 in the spleen of SeMet-supplemented mice were lower than those in control mice. The mRNA expression level of a Th1 cytokine, interferon (IFN)-γ, in the spleen of SeMet-supplemented mice was higher than that in control mice. Splenocytes restimulated with OVA in vitro from SeMet-supplemented mice produced lower amounts of IL-4 and IL-13 than those of control mice and higher amounts of IFN-γ than those from the control mice. These results suggest that oral SeMet supplementation suppresses OVA-induced ACA reaction by lowered Th2 cytokine production and augmenting Th1 cytokine production.

Vigna angularis inhibits mast cell-mediated allergic inflammation.

The aim of the present study was to elucidate whether extracts of Vigna angularis (EVA) inhibit allergic inflammatory reactions and to elucidate the possible mechanisms of action. For the assessment of allergic inflammatory response, histamine release and the expression of pro-inflammatory cytokines from human mast cells (HMC-1) were examined. To identify the underlying mechanisms of action, intracellular calcium and the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) were assayed. To confirm the effects of EVA in vivo, systemic and local allergic reaction mouse models were employed. EVA dose-dependently reduced phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced histamine release from mast cells. The inhibitory effects of EVA on the release of histamine from mast cells were mediated by the reduction of intracellular calcium levels. EVA decreased the PMACI-stimulated gene expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. The inhibitory effects of EVA on pro-inflammatory cytokines were NF-κB- and MAPK-dependent. In addition, EVA inhibited compound 48/80-induced systemic anaphylaxis and immunoglobulin E (IgE)-mediated cutaneous anaphylaxis. Our findings provide evidence that EVA inhibits mast cell-derived allergic inflammation, and suggest the possible therapeutic application of EVA in allergic inflammatory disorders.

Resveratrol inhibits IgE-mediated basophilic mast cell degranulation and passive cutaneous anaphylaxis in mice.

Resveratrol is a phytoalexin abundantly found in red grape skin and is effective in antitumor and antiinflammation associated with immune responses. This study investigated whether resveratrol suppressed immunoglobulin (Ig)E-mediated allergic responses and passive cutaneous anaphylaxis (PCA) in rat RBL-2H3 mast cells and in BALB/c mice. The release of ß-hexosaminidase and histamine was enhanced in mast cells sensitized with anti-dinitrophenyl (DNP)-IgE and subsequently stimulated by DNP-human serum albumin (HSA), indicative of mast cell degranulation. When mast cells were pretreated with nontoxic resveratrol at 1-25 µmol/L, such induction was dose dependently diminished. Spleen tyrosine kinase (Syk) and phospholipase Cγ (PLCγ) of sensitized mast cells were activated by stimulation with DNP-HSA antigen, which was dampened by ≥5 µmol/L resveratrol. The phosphorylation of protein kinase C (PKC)µ and PKCθ was attenuated by administering resveratrol to DNP-HSA-exposed mast cells, whereas quiescent PKCζ/λ in sensitized cells was dose-dependently activated by resveratrol. Male BALB/c mice were sensitized for 24 h with DNP-IgE and orally administered with resveratrol 1 h before the DNP-HSA challenge. The histamine concentration was enhanced in sensitized mice challenged to DNP-HSA, which was reversed by administration of 10 mg/kg resveratrol. Additionally, it encumbered the tissue activation of Syk, PLCγ, and PKCµ in antigen-exposed mice. Resveratrol decreased IgE-mediated PCA and alleviated allergic edema of mouse ear and dorsal skin. Mast cell degranulation and allergic inflammation, accompanying the induction of monocyte chemotactic protein-1 and macrophage inflammatory protein-2, were inhibited by supplementing resveratrol to antigen-challenged mice. Resveratrol inhibited mast cell-derived, immediate-type allergic reactions, and these responses of resveratrol suggest possible therapeutic strategies in preventing allergic inflammatory diseases.

Inula japonica extract inhibits mast cell-mediated allergic reaction and mast cell activation.

ETHNOPHARMACOLOGICAL RELEVANCE: The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for the treatment of bronchitis, digestive disorders, and inflammation. However, the mechanisms underlying its anti-inflammatory effects remain yet to be elucidated. The objectives of this study were 1) to assess the anti-allergic activity of the ethanol extract of flowers of Inula japonica extract (IFE) in vivo, 2) to investigate the mechanism of its action on mast cells in vitro, and 3) to identify its major phytochemical compositions. MATERIALS AND METHODS: The anti-allergic activity of IFE was evaluated using mouse bone marrow-derived mast cells (BMMCs) in vitro and a passive cutaneous anaphylaxis (PCA) animal model in vivo. The effects of IFE on mast cell activation were evaluated in terms of degranulation, eicosanoid generation, Ca(2+) influx, and immunoblotting of various signaling molecules. RESULTS: IFE inhibited degranulation and the generation of eicosanoids (PGD(2) and LTC(4)) in stem cell factor (SCF)-stimulated BMMCs. Biochemical analysis of the SCF-mediated signaling pathways demonstrated that IFE inhibited the activation of multiple downstream signaling processes including mobilization of intracellular Ca(2+) and phosphorylation of the mitogen-activated protein kinases (MAPKs), PLCγ1, and cPLA(2) pathways. When administered orally, IFE attenuated the mast cell-mediated PCA reaction in IgE-sensitized mice. Its major phytochemical composition included three sesquiterpenes, 1-O-acetylbritannilactone, britanin and tomentosin. CONCLUSIONS: This study suggests that IFE modulates eicosanoids generation and degranulation through the suppression of SCF-mediated signaling pathways that would be beneficial for the prevention of allergic inflammatory diseases. Anti-allergic activity of IFE may be in part attributed particularly to the presence of britanin and tomentosin as major components evidenced by a HPLC analysis.

[Real-time detection of mast cell degranulation in anaphylactoid reaction].

OBJECTIVE: To establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction. METHOD: A CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells. RESULT: Before antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1). CONCLUSION: Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.

TCM grammar systems: an approach to aid the interpretation of the molecular interactions in Chinese herbal medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Interpreting the molecular interactions in Chinese herbal medicine will help to understand the molecular mechanisms of Traditional Chinese medicines (TCM) and predict the new pharmacological effects of TCM. Yet, we still lack a method which could integrate the concerned pieces of parsed knowledge about TCM. MATERIALS AND METHODS: To solve the problem, a new method named TCM grammar systems was proposed in the present article. The possibility to study the interactions of TCM at the molecular level using TCM grammar systems was explored using Herba Ephedrae Decoction (HED) as an example. RESULTS: A platform was established based on the formalism of TCM grammar systems. The related molecular network of Herba Ephedrae Decoction (HED) can be extracted automatically. The molecular network indicates that Beta2 adrenergic receptor, Glucocorticoid receptor and Interleukin12 are the relatively important targets for the anti-anaphylaxis asthma function of HED. Moreover, the anti-anaphylaxis asthma function of HED is also related with suppressing inflammation process. The results show the feasibility using TCM grammar systems to interpret the molecular mechanism of TCM. Although the results obtained depend on the database absolutely, recombination of existing knowledge in this method provides new insight for interpreting the molecular mechanism of TCM. CONCLUSIONS: TCM grammar systems could aid the interpretation of the molecular interactions in TCM to some extent. Moreover, it might be useful to predict the new pharmacological effects of TCM. The method is an in silico technology. In association with the experimental techniques, this method will play an important role in the understanding of the molecular mechanisms of TCM.

Insamhodo-tang, a traditional Korean medicine, regulates mast cell-mediated allergic inflammation in vivo and in vitro.

AIM OF THE STUDY: Insamhodo-tang (IHT) has traditionally been used in Korea to treat a variety of diseases, including chronic cough, tuberculosis, and chronic bronchitis. However, the anti-allergic and anti-inflammatory effects of IHT and its molecular mechanisms have yet to be clearly elucidated. In this study, we attempted to evaluate the effects of IHT on mast cell-mediated allergy inflammation in vitro and in vivo. MATERIALS AND METHODS: We investigated to ascertain the pharmacological effects of IHT on both compound 48/80-induced and 2,4-dinitrofluorobenzene (DNFB)-induced allergic reactions under in vivo conditions. Additionally, to find a possible explanation for the anti-inflammatory mechanisms of IHT, we evaluated the regulatory effects of IHT on the level of inflammatory mediators in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cells (HMC-1). RESULTS: The finding of this study demonstrated that IHT reduced compound 48/80-induced systemic anaphylactic shock, DNFB-induced dermatitis, and ear swelling responses in mice. Additionally, IHT inhibited the production of interleukin (IL)-6, IL-8, and TNF-α, as well as the activation of nuclear factor-κB and caspase-1 in PMACI-stimulated HMC-1. CONCLUSION: Collectively, the findings of this study provide us with a novel insight into the pharmacological actions of IHT as a potential molecule for use in the treatment of allergic inflammation diseases.

Silibinin attenuates mast cell-mediated anaphylaxis-like reactions.

Silibinin is known to have hepatoprotective, anti-carcinogenic and anti-inflammatory effects. However, roles of silibinin in the immediate-type allergic reactions (anaphylaxis) have not fully been investigated. In the present study, we have demonstrated that silibinin attenuated mast cell-mediated anaphylaxis-like reactions involved in allergic diseases. Oral administration of silibinin inhibited compound 48/80-induced passive cutaneous anaphylaxis-like reaction in mice. Silibinin also attenuated anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-mediated passive systemic and cutaneous anaphylaxis. Silibinin had no cytotoxicity on rat peritoneal mast cells (RPMC). Silibinin dose-dependently reduced histamine release from RPMC activated by compound 48/80 or anti-DNP IgE. Moreover, silibinin inhibited the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 in RPMC. Pretreatment of silibinin suppressed the antigen-stimulated calcium uptake and activation of nuclear factor-kappa B (NF-kappaB) in RPMC. Furthermore, silibinin increased the intracellular cAMP level. Increased cAMP, decreased calcium uptake and suppressed NF-kappaB activity might be involved in the inhibitory effect of silibinin on the secretory response. Our findings provide possibility that silibinin may serve as an effective therapeutic agent for allergic diseases.