Β-hexosaminidase Release Research Articles

Aurora kinase inhibitor tozasertib suppresses mast cell activation in vitro and in vivo.

Mast cells are important in allergic reactions. Here, we assessed the anti-allergic effects of the anti-cancer drug tozasertib specifically regarding regulatory effects on mast cell activation. Tozasertib effects on mast cell degranulation were determined by measuring β-hexosaminidase and histamine release and by assessing morphological changes in RBL-2H3 and mouse bone marrow-derived mast cells (BMMCs) stimulated with mouse anti-dinitrophenyl (DNP)-IgE/DNP-human serum albumin or human LAD2 cells activated with phorbol-12-myristate 13-acetate plus calcium ionophore (PMACI). Western blots were performed to detect the expression of molecules involved in NF-κB, MAPK, and Aurora kinase signalling. in vivo anti-allergic effects of tozasertib were determined in the murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) models. Tozasertib treatment decreased high-affinity IgE receptor (FcεRI) or PMACI-mediated degranulation in RBL-2H3 cells and in BMMCs or LAD2 cells as shown by β-hexosaminidase or histamine levels. Similarly, tozasertib prevented morphological changes in mast cells, such as particle release and F-actin reorganization. In addition, tozasertib markedly decreased expression of phosphorylated (p)-NF-κB p65, p-Erk1/2, p-p38, and p-Aurora A/B, indicating that tozasertib can inhibit the signalling pathway mediating mast cell activation. Tozasertib attenuated IgE/Ag-induced PCA dose-dependently, as shown by reduced Evans blue staining. Similarly, tozasertib reduced body temperature levels and serum histamine levels in OVA-challenged ASA mice. The Aurora kinase inhibitor tozasertib suppressed mast cell activation in vitro and in vivo. Tozasertib may be a potential drug, targeting mast cell activation, to treat allergic diseases or mastocytosis.

TAK1ng aim at mechanisms of allergic inflammation: contribution of TAK1 activity in allergen‐mediated mast cell activation

Allergic inflammatory disorders are at epidemic levels worldwide. Mast cells drive this inappropriate immune response via release of a variety of pro‐inflammatory mediators in response to environmental cues detected by the IgE‐FcɛRI complex, but the contributing molecular mechanisms are not fully understood. Transforming growth factor β‐activated kinase 1 (TAK1) is a known participant in related signaling through both the MAPK and NFκB pathways; however, the role of TAK1 in IgE‐FcɛRI mediated signaling and resultant allergic inflammation remains unknown. This study was carried out to assess the role of TAK1 in IgE‐mediated mast cell activation and to determine the effect its inhibition would have on the severity of an allergic inflammatory response. Assessment of the role of TAK1 was conducted by exploiting the characterized inhibitory function of 5Z‐7‐oxozeaenol (OZ), a resorcylic acid lactone established to be an ATP‐competitive selective inhibitor of TAK1. Bone marrow‐derived mast cells were sensitized with allergen‐specific IgE and treated with the corresponding allergen for various times in the presence or absence of OZ. Mast cell signaling was measured by western blotting, induced gene expression by qPCR, pro‐inflammatory mediator release by ELISA, mast cell degranulation by β‐hexosaminidase release assay, and calcium mobilization alterations via Indo‐1 based spectrofluorometry. We detect novel activation of TAK1 at Ser412 in response to IgE‐mediated activation under SCF‐c‐kit potentiation in a mast cell‐driven response characteristic of allergic inflammation, which is potently blocked by TAK1 inhibitor OZ. We therefore interrogated the role of TAK1 in a series of mast cell mediator responses using IgE‐sensitized murine bone marrow‐derived mast cells, stimulated with allergen under several TAK1 inhibition strategies. TAK1 inhibition resulted in significant impairment in the phosphorylation of MAPKs p38, ERK, and JNK; and mediation of the NFκB pathway via IκBα. Impaired gene expression and near abrogation in release of pro‐inflammatory cytokines TNF (p<0.0001), IL‐6 (p<0.0001), IL‐13 (p<0.0001), and chemokines CCL1 (p=0.0002), and CCL2 (p<0.0001) was detected. Finally, a significant inhibition of mast cell degranulation (p<0.0001), accompanied by an impairment in calcium mobilization, was observed in the TAK1‐inhibited cells. These results suggest that TAK1 acts as a signaling node, not only linking the MAPK and NFκB pathways in driving the late‐phase response, but also initiation of the degranulation mechanism of the mast cell early‐phase response following allergen recognition and may warrant consideration in future therapeutic development.Support or Funding InformationFunding: This work was supported by the Natural Sciences and Engineering Research Council (NSERC), the Canada Foundation for Innovation (CFI), the Ontario Research Fund (ORF), and Ontario Graduate Scholarships.

Discovery and analysis the anti-pseudo-allergic components from Perilla frutescens leaves by overexpressed MRGPRX2 cell membrane chromatography coupled with HPLC-ESI-IT-TOF system.

Screen and identify the anti-pseudo-allergic activity components of Perilla frutescens leaves that interacted with MRGPRX2 (a new reported pseudo-allergic reaction-related receptor). An overexpressed MRGPRX2 cell membrane chromatography (CMC) coupled with HPLC-ESI-IT-TOF system has been established to screen and identify the effective components from P.frutescens leaves. A frontal analysis method was performed to investigate the binding affinity between ligands and MRGPRX2. Their activity of relieving pseudo-allergic reaction was evaluated in vitro by histamine release assay, β-hexosaminidase release assay and intracellular Ca2+ mobilization assay. Extract of P.frutescens leaves was proved to be effective in anti-pseudo-allergic reaction by inhibiting MRGPRX2. Apigenin (API) and rosmarinic acid (ROS) were confirmed to be the potential anti-allergy compounds that could bind with MRGPRX2. The binding affinity (KD ) of ROS and API with MRGPRX2 was (8.79±0.13)×10-8 m and (6.54±1.69)×10-8 m, respectively. The IC50 of API inhibiting laboratory of allergic disease 2 cells degranulation was also determined to be (51.96±0.18)μm. A MRGPRX2/CMC coupled with HPLC-ESI-IT-TOF system was successfully established and applied to discover the effective components from P.frutescens leaves.

Qing-Kai-Ling Injection Induces Immediate Hypersensitivity Reaction via the Activation of Anaphylatoxin C3.

Background and Objective: Qing-Kai-Ling (QKL) is derived from a famous ancient Chinese patent medicine Angong Niuhuang pills (ANP) which has been used across Asia, especially in China, for the treatment of “febrile disease,” such as stroke, encephalitis and meningitis for hundreds of years. As an extract of ANP without heavy metal, the clinical applicability of QKL is more intensive, of which its injection is commonly used in acute and serious diseases. This study aims to clarify the potential mechanisms of immediate hypersensitivity reaction (IHR) induced by QKL injection (QKLI). Methods: β-hexosaminidase release assay was performed on the human mast cell line LAD2 and mouse peritoneal mast cells. T helper 2 (Th2) immunity-amplified mice were prepared by aluminum adjuvant. Anaphylactic shock was detected by measuring rectal thermometry in propranolol-pretreated mice. For evaluating microvascular permeability, Evans Blue extravasation assay was used. Serum total IgE (tIgE) and the activated complement-derived anaphylatoxin C3 (C3a) levels were measured by ELISA. Results: QKLI was unable to elevate serum tIgE level in the Th2 immunity-amplified mice, but can increase vasopermeability and trigger anaphylaxis after the first injection. By screening seven fractions of QKLI, only the extract of Isatidis Radix (Isatis tinctoria L.) induced hindpaw Evans Blue extravasation, which was disappeared in Isatidis Radix-free QKLI. Mechanism study indicated that QKLI or Isatidis Radix-caused IHR could be blocked by the antagonists for histamine or C3a, rather than PAF or C5a. Consistently, QKLI and Isatidis Radix could also directly activate human serum complement-derived anaphylatoxin 3 (C3) in vitro with the half effective concentration values of 0.69% and 218.6 μg/ml, respectively. Conclusion: QKLI-IHR is complement activation-related pseudoallergy, rather than an IgE-mediated allergy. QKLI activates C3 and might consequently provoke mast cells to release histamine, which is a principal effector of its IHR. The pseudoallergic reaction induced by QKLI was attributed to the extract of Isatidis Radix. This study suggests a potential therapeutic strategy for the prophylaxis and treatment of QKLI-IHR.

The role of myricetin from Rhodomyrtus tomentosa (Aiton) Hassk fruits on downregulation of FcɛRI-mediated mast cell activation.

Rhodomyrtus tomentosa was reported to contain various bioactive metabolites, especially phenolic compounds. In the present study, the suppressive activity of phenolic compound from R. tomentosa fruits on mast cell activation was investigated in vitro. The result showed that myricetin was isolated from R. tomentosa fruits and its characterization was identified by nuclear magnetic resonance spectroscopy. Notably, myricetin was found to be effective in inhibition of mast cell degranulation by attenuating the release of β-hexosaminidase and the elevation of intracellular calcium. Moreover, myricetin exhibited inhibitory effect on the production of IL-4 and Tumor necrosis factor alpha (TNF-α) in a concentration-dependent manner. Furthermore, high antioxidant activity of myricetin due to scavenging 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and ABTS+ radicals was also evidenced. Notably, the activation of FcɛRI-mediated signaling molecules including Syk, PLCγ, and NF-κB was also suppressed by myricetin treatment. Accordingly, myricetin from R. tomentosa fruits could be suggested as a functional food for the amelioration of allergic diseases. PRACTICAL APPLICATIONS: Polyphenol have been shown to exert various biological activities and health beneficial effects. Results from the present study revealed that myricetin from R. tomentosa fruits possesses the inhibitory effect on allergic response in mast cells. Therefore, myricetin from R. tomentosa fruits could be developed as a functional ingredient for the amelioration of allergic diseases.

Use of Engineered Nanoparticles (ENPs) for the Study of High-Affinity IgE FcεRI Receptor Engagement and Rat Basophilic Leukemia (RBL) Cell Degranulation.

Degranulation of mast cells and basophils occurs after the cross-linking of FcεRI receptor-bound IgE by multivalent allergens, resulting in the release of a range of de novo synthesized and preformed mediators of the allergic response. β-Hexosaminidase release is usually measured as a simple readout for degranulation. Furthermore, the rat basophilic leukemia (RBL)-2H3 cell line is commonly used for measuring degranulation, monitoring β-hexosaminidase release. Here, we describe surface-engineered and modified nanoparticles with specific ligands in order to study the signaling and cellular responses of the RBL-2H3 cell line.

Enzymatically synthesized glycogen inhibited degranulation and inflammatory responses through stimulation of intestine.

The patients of type I allergic diseases were increased in the developed countries. Recently, many studies have focused on food factors with anti-allergic activities. Enzymatically synthesized glycogen, a polysaccharide with a multi-branched α-1,4 and α-1,6 linkages, is a commercially available product from natural plant starch, and has immunostimulation activity. However, effect of enzymatically synthesized glycogen on the anti-allergic activity was unclear yet. In this study, we investigated that enzymatically synthesized glycogen inhibited allergic and inflammatory responses using a co-culture system consisting of Caco-2 and RBL-2H3 cells. Enzymatically synthesized glycogen inhibited antigen-induced β-hexosaminidase release and production of TNF-α and IL-6 in RBL-2H3 cells in the co-culture system. Furthermore, enzymatically synthesized glycogen inhibited antigen-induced phosphorylation of tyrosine kinases, phospholipase C γ1/2, mitogen-activated protein kinases and Akt. Anti-allergic and anti-inflammatory activities of enzymatically synthesized glycogen were indirect action through stimulating Caco-2 cells, but not by the direct interaction with RBL-2H3 cells, because enzymatically synthesized glycogen did not permeate Caco-2 cells. These findings suggest that enzymatically synthesized glycogen is an effective food ingredient for prevention of type I allergy through stimulating the intestinal cells.

Quercitrin, the Main Compound in Wikstroemia indica, Mitigates Skin Lesions in a Mouse Model of 2,4‐Dinitrochlorobenzene‐Induced Contact Hypersensitivity

Hapten-induced contact hypersensitivity (CHS) is widely utilized to induce immune activation in animal models of allergic contact dermatitis. Our previous findings suggested that the 95% EtOH extract of Wikstroemia indica (L.) C. A. Mey. has antiallergic and anti-inflammatory effects in DNCB-treated CHS SKH-1 hairless mice. The aim of this study was to evaluate the protective effects of compounds isolated from the EtOAc fraction of W. indica in RBL-2H3 cells and 2,4-dinitrochlorobenzene- (DNCB-) induced CHS mice. Of eight compounds in W. indica, that is, umbelliferone, daphnoretin, wikstrocoumarin, (+)-syringaresinol, tricin, (+)-lariciresinol, erythro-guaiacylglycerol-β-coniferyl ether, and quercitrin, quercitrin exhibited the most antiallergic activity against antigen-induced β-hexosaminidase release and IL-4 mRNA expression, which are markers of degranulation in RBL-2H3 cells. After a 7-sensitizing period, 14 days of DNCB treatment with or without topical pimecrolimus (1%) or quercitrin (0.5%) treatment, quercitrin was found to suppress DNCB-induced increases in serum IL-4 and IgE concentrations and transepidermal water loss. These results indicate that quercitrin has therapeutic potential for treatment of allergies and allergy-related contact dermatitis.

Trigonelline: An alkaloid with anti-degranulation properties

Trigonelline, one of the alkaloids contained in coffee, is important not only as one of the constituents of aroma and flavor in coffee but also as a useful source of nutrition. Its anti-microbial, anti-carcinogenic, and anti-hyperglycemic effects have been investigated in previous studies. However, there have not been any studies examining the anti-degranulation effect of trigonelline. In this study, the anti-degranulation effect of trigonelline was evaluated in in vitro and in vivo models using a rat basophilic leukemia cell line, RBL-2H3 cells, and a passive cutaneous anaphylaxis (PCA) reaction in mice, respectively. In the β-hexosaminidase release assay, trigonelline effectively suppressed antigen-induced degranulation of RBL-2H3 cells in a dose-dependent manner without cytotoxicity. Trigonelline also inhibited FcεRI-mediated intracellular signaling pathways, such as phosphorylation of PLCγ1, PI3 K, and Akt, in antigen-stimulated RBL-2H3 cells and suppressed the PCA response in mice. Moreover, trigonelline also inhibited the microtubule formation in RBL-2H3 cells, indicating that trigonelline could inhibit IgE-sensitized mast cell degranulation by attenuating both the intracellular calcium-dependent and independent pathways. These results revealed that trigonelline possesses the anti-degranulation effect against the development of allergic diseases.

Anti-Atopic Effect of Acorn Shell Extract on Atopic Dermatitis-Like Lesions in Mice and Its Active Phytochemicals.

To investigate the potential effects of acorn shells on atopic dermatitis (AD), we utilized oxazolone (OX)- or 2,4-dinitrochlorobenzene (DNCB)-induced AD-like lesion mouse models. Our research demonstrates that Acorn shell extract (ASE) improved the progression of AD-like lesions, including swelling, which were induced by oxazolone on Balb/c mouse ears. Additionally, ASE significantly decreased the ear thickness (OX: 0.42 ± 0.01 mm, OX-ASE: 0.32 ± 0.02 mm) and epidermal thickness (OX: 75.3 ± 32.6 µm, OX-ASE: 46.1 ± 13.4 µm). The continuous DNCB-induced AD mouse model in SKH-1 hairless mice demonstrated that ASE improved AD-like symptoms, including the recovery of skin barrier dysfunction, Immunoglobulin E hyperproduction (DNCB: 340.1 ± 66.8 ng/mL, DNCB-ASE: 234.8 ± 32.9 ng/mL) and an increase in epidermal thickness (DNCB: 96.4 ± 21.9 µm, DNCB-ASE: 52.4 ± 16.3 µm). In addition, we found that ASE suppressed the levels of AD-involved cytokines, such as Tumor Necrosis Factor α, IL-1β, IL-25 and IL-33 in both animal models. Furthermore, gallic acid and ellagic acid isolated from ASE suppressed β-hexosaminidase release and IL-4 expression in RBL-2H3 cells. The acorn shell and its active phytochemicals have potential as a new remedy for the improvement of atopic dermatitis and other inflammatory diseases.

Inhibition of Calcineurin and Glycogen Synthase Kinase-3β by Ricinoleic Acid Derived from Castor Oil.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+ -signal transduction pathway-mediated cell cycle regulation in a yeast-based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+ -signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast-based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth-restoration activity at 5 μg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 μM in a substrate-competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring β-hexosaminidase (β-HEX) release in RBL-2H3 cells. RA at 50 μM suppressed the release of β-HEX from RBL-2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase-3β (GSK-3β), as determined by a kinase assay using recombinant human GSK-3β. RA inhibited GSK-3β at Ki = 1.43 μM in a peptide substrate-competitive manner. The inhibition of GSK-3β by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 μM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

Variegatic acid from the edible mushroom Tylopilus ballouii inhibits TNF-α production and PKCβ1 activity in leukemia cells

Variegatic acid, isolated from Tylopilus ballouii dry fruiting bodies, is an inhibitor of β-hexosaminidase release and tumor necrosis factor (TNF)-α secretion from rat basophilic leukemia (RBL-2H3) cells, with IC50 values of 10.4 μM and 16.8 μM, respectively. On the other hand, it inhibits PKCβ1 activity with an IC50 value of 36.2 μM.

Influence of Cyclophosphamide on L-Asparaginase-Induced Allergy in Animal Model

BACKGROUND: L-Asparaginase (L-ASP) is one of the essential drugs for acute lymphoblastic leukemia and lymphoma. Since L-ASP proteins used clinically are derived from bacteria, they often cause allergies, including anaphylaxis. However, the characteristics of L-ASP-induced hypersensitivity are not well documented so far. Also, there is neither suitable treatment for hypersensitivity to L-ASP nor adequate experimental model of L-ASP allergy. In addition, some anti-cancer drugs, used concomitantly with L-ASP, are known to affect lymphocyte activities, although the role of such drugs on L-ASP hypersensitivity is not known. Previously, we have established mouse model of L-ASP allergy and found that a neutralizing antibody (Ab) of IgE is effective to inhibit L-ASP-induced allergic reaction. Aims: In order to establish in vitro model of L-ASP allergy, we used RBL-2H3 mast cells and evoked degranulation of the cells sensitized with L-ASP-allergic mouse serum using L-ASP as an antigen. Furthermore, we examined the effect of cyclophosphamide (CY), which reportedly impairs the functions of regulatory T (Treg) cells, on L-ASP allergy both in vivo and in vitro. Methods: Male BALB/c mice (7 week old) were sensitized with L-ASP (100 units) with Al(OH)3 gel (1 mg) on days 1 and 15. The right ears of the mice were locally sensitized on days 18, 21, 24 by i.d. injection of L-ASP (10 units). Antigen challenge was carried out on day 27 by i.d. injection (10 units). CY (75, 150, 300 mg/kg) was i.p. administrated at day -1 and day 13. Anti-IgE treatment (BD Bioscience, clone R35-92; 100 μg, i.p.) was carried out on the day before L-ASP challenge. The serum was collected on day 27. Total IgE level in the serum was measured by ELISA kit (Yamasa). RBL-2H3 cells were sensitized by the serum and stimulated by L-ASP to determine β-hexosaminidase (β-Hex) release in vitro. Results : L-ASP-sensitization induced allergic ear edema and an increase in serum IgE level in mice, both of which are augmented by an administration of CY at 150 mg/kg. L-ASP-induced allergic ear edema was inhibited by a pretreatment with anti-IgE Ab. L-ASP-induced CY-enhanced ear edema was also inhibited by anti-IgE Ab. On the other hand, at 300 mg/kg of CY, L-ASP-induced ear edema was much lower than 150 mg/kg group, though IgE concentration in the serum was as high as CY 150 mg/kg group. When RBL-2H3 cells were sensitized by anti-L-ASP serum, L-ASP challenge induced β-Hex release. Anti-L-ASP serum of CY 150 mg/kg-treated mice induced higher β-Hex release than normal anti-L-ASP sera in vitro, though that of CY 300 mg/kg-treated mice did not induce β-Hex release. After removing IgG from the serum of CY 300 mg/kg-treated mice using protein G Mag Sepharose (GE Healthcare), β-Hex release became higher than normally sensitized mice. Anti-IgE Ab treatment both ex vivo and in vitro inhibited L-ASP-induced β-Hex release from the RBL-2H3 cells sensitized by anti-L-ASP serum of CY-treated mice. Conclusions: In this study, we showed that L-ASP sensitization induced IgE production in vivo and this serum could bring about β-Hex release from RBL-2H3 cells in vitro; both allergic reactions were inhibited by an anti-IgE antibody which binds to Fc region of IgE molecule. From these results, we propose that non-anaphylactogenic neutralizing antibody of IgE, such as omalizumab, can be a candidate drug for the treatment of L-ASP hypersensitivity. Also in this study, CY induced biphasic effects on L-ASP allergy in mice, enhancing it at low dosage and attenuating it at high dosage. Since CY is reportedly impair the functions of Treg cells, CY-induced suppression of Treg may enhance Th2 responses so as to augment L-ASP hypersensitivity. Further investigations for the detection and avoidance of L-ASP hypersensitivity are under the way. Disclosures No relevant conflicts of interest to declare.

Chamaejasmine Isolated from Wikstroemia dolichantha Diels Suppresses 2,4-Dinitrofluoro-benzene-Induced Atopic Dermatitis in SKH-1 Hairless Mice.

Plants of the genus Wikstroemia have long been used as traditional medicines to treat diseases like pneumonia, rheumatism, and bronchitis. This study was designed to determine the effect of chamaejasmine, a biflavonoid present in W. dolichantha, on atopic dermatitis (AD)-like skin lesions in a 2,4-dinitrochlorobenzene (DNCB)-induced murine model of AD. Initially, we examined the anti-allergic activities of ten flavonoids from W. dolichantha by measuring β-hexosaminidase release from RBL-2H3 cells. Subsequently, an SKH-1 hairless mouse model of AD was developed based on the topical application of DNCB. Chamaejasmine (0.5%) or pimecrolimus (1%, positive control) were applied to dorsal skins of DNCB-sensitized AD mice for two weeks. Serum IL-4 and IgE levels were determined using enzyme-linked immunosorbent assay kits and transepidermal water loss (TEWL) and skin hydration were measured using a Tewameter TM210 and a SKIN-O-MAT, respectively. Of the ten flavonoids isolated from W. dolichantha, chamaejasmine most potently inhibited DNP-specific IgE-induced degranulation in RBL-2H3 cells. Topical administration of chamaejasmine attenuated the clinical symptoms of DNCB-induced dermatitis (i.e., itching, dryness, erythema, and edema). Histological analyses demonstrated that dermal thickness and mast cell infiltration in dermis were significantly reduced by chamaejasmine. In addition, 0.5% chamaejasmine inhibited DNCB-induced increases in total IL-4 and IgE levels in serum, improved skin barrier function, and increased epidermis moisture. Our findings suggest chamaejasmine might be an effective therapeutic agent for the treatment of atopic diseases.

Anti-Inflammatory Effect of Essential Oils Extracted from Wood of Four Coniferous Tree Species

The aim of this study was to evaluate the anti-inflammatory effects of essential oils extracted from the wood of Chamaecyparis obtusa, Pinus densiflora, Pinus koraiensis, and Larix kaempferi. Essential oils were extracted by hydrodistillation, and their chemical components were determined by GC/MS. Major chemical components of these essential oils were α-cadinol (19.25%), τ-muurolol (14.20%), and α-pinene (13.74%) in C. obtusa; α-pinene (47.16%), longifolene (14.31%), β-phellandrene (11.78%), and β-pinene (11.02%) in P. densiflora; α-pinene (13.49%) and longifolene (10.79%) in P. koraiensis, and geranyl linalool (23.58%) and α-pinene (18.57%) in L. kaempferi. To evaluate the anti-inflammatory effects of essential oils, lipopolysaccharide (LPS)-induced RBL-2H3 mast cells were treated with these essential oils; then, the changes in the mRNA expression level of the cytokines IL-4 and IL-13 were examined. Further, degranulation was evaluated by measuring β-hexosaminidase release. After LPS-induced RBL-2H3 mast cells were exposed to 10−7% of all types of essential oils, the gene expression levels of IL-4 and IL-13 within the cells remarkably decreased. The relative mRNA expression level of IL-4 was 69.6% in P. densiflora, 63.2% in P. koraiensis, 55.1% in C. obtusa, and 45.8% in L. kaempferi compared with that in the group treated with LPS. The mRNA expression level of L-13 should a similar trend. The inhibitory rate of IL-13 mRNA expression of P. densiflora, P. koraiensis, C. obtusa, and L. kaempferi was 57.8%, 57.1%, 51.1%, and 34.5%, respectively. β-Hexosaminidase release significantly decreased following the treatment with the four types of essential oils. The rate of β-hexosaminidase release were 38.1% C. obtusa; 33.0% P. densiflora; 27.4% P. koraiensis; and 9.1% L. kaempferi. Among all types of essential oils, that extracted from P. densiflora wood showed the highest anti-inflammatory activity. These results show that the tested essential oils exert an anti-inflammatory effect through the inhibition of degranulation and expression of cytokines.

Design, synthesis and biological activity of a novel ethylenediamine derivatives as H1 receptor antagonists

In this study, a series of novel ethylenediamine compounds were obtained by structural modification of the lead compounds with thonzylamine, and using the principle of modifying by bioisostere formation and modification with alkyl groups. In vitro assay, the biological activities showed that the target compounds have good properties in inhibiting mast cell degranulation and releasing histamine and β-aminohexidase, such as the compounds 5c, 5g, 5k, 5l and 5o, especially of compound 5k to mast cell degranulation is IC50 = 0.0106 ± 0.001 μmol⋅L−1, histamine release was IC50 = 0.0192 ± 0.005 μmol⋅L−1 and β-hexosaminidase release was IC50 = 0.0455 ± 0.002 μmol⋅L−1in vitro. At the same time, in vivo biological activities assay results showed that have a good Histamie induce bronchospasm effect with relatively long duration and good protective effect in vivo, among which the protective effect of compound 5k was 79.74 ± 0.30%, compounds 5c, 5g, 5k, 5l and 5o could inhibit the capillary permeability of increasing which were caused by histamine.

Amphiphilic Triazine Polymer Derivatives as Antibacterial And Anti-atopic Agents in Mice Model

Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.

3-Benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one suppresses FcεRI-mediated mast cell degranulation via the inhibition of mTORC2-Akt signaling

Mast cells express high-affinity IgE receptor (FcεRI) on their surface, cross-linking of which leads to the immediate release of proinflammatory mediators such as histamine but also late-phase cytokine secretion, which are central to the pathogenesis of allergic diseases. Despite the growing evidences that mammalian target of rapamycin (mTOR) plays important roles in the immune system, it is still unclear how mTOR signaling regulates mast cell function. In this study, we investigated the effects of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as an mTOR agonist on FcεRI-mediated allergic responses of mast cells. Our data showed that administration of 3BDO decreased β-hexosaminidase, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI cross-linking, which was associated with an increase in mTOR complex 1 (mTORC1) signaling but a decrease in activation of Erk1/2, Jnk, and mTORC2-Akt. In addition, we found that a specific Akt agonist, SC79, is able to fully restore the decrease of β-hexosaminidase release in 3BDO-treated BMMCs but has no effect on IL-6 release in these cells, suggesting that 3BDO negatively regulates FcεRI-mediated degranulation and cytokine release through differential mechanisms in mast cells. The present data demonstrate that proper activation of mTORC1 is crucial for mast cell effector function, suggesting the applicability of the mTORC1 activator as a useful therapeutic agent in mast cell-related diseases.

Effect of Rhus verniciflua Extract on IgE-Antigen-Mediated Allergic Reaction in Rat Basophilic Leukemic RBL-2H3 Mast Cells and Passive Cutaneous Anaphylaxis in Mice.

Rhus verniciflua is widely known for its antioxidant, antibacterial, anticancer, and antiaging efficacy and α-glucosidase inhibition. This study was designed whether Rhus verniciflua extracts inhibit the IgE-antigen-mediated allergic reaction in RBL-2H3 mast cells, and it further investigated the FcεRI- and arachidonate-signaling by which Rhus verniciflua extracts exert its antiallergic effects. IgE-antigen-sensitized RBL-2H3 mast cells were investigated for the cytotoxicity of Rhus verniciflua extracts and β-hexosaminidase release, and inflammatory mediators (e.g., TNF-α, IL-4, IL-6, histamine, and PGD2) were then assessed. Additionally, we examined expressions of genes involved in arachidonate- and FcεRI-signaling pathway in RBL-2H3. Rhus verniciflua extracts inhibited β-hexosaminidase release and production of the inflammatory mediators in RBL-2H3. Rhus verniciflua extracts reduced amounts of histamine and expressions of FcεRI signaling-related genes such as Lyn and Syk and phosphorylation of extracellular signal-regulated kinase in mast cells. Finally, in late allergic responses, Rhus verniciflua extracts reduced PGD2 release and COX-2 and cPLA2 phosphorylation expressions from IgE-antigen-mediated mast cells. Lastly, 250–500 mg/kg RVE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergy properties of Rhus verniciflua extracts in FcɛRI-mediated allergic reaction.

Exposure to silver nanoparticles primes mast cells for enhanced activation through the high-affinity IgE receptor

Mast cells are a key effector cell in type I allergic reactions. It has been shown that environmental exposures such as diesel exhaust and heavy metals exacerbate mast cell degranulation and activation. Today, the use of engineered nanomaterials (ENMs) is rapidly expanding and silver nanoparticles (AgNP) are one of the mostly widely utilized ENMs, primarily for their antimicrobial properties, and are being incorporated into many consumer and biomedical products. We assessed whether pre-exposure of bone marrow-derived mast cells (BMMCs) to 20 nm AgNPs enhanced degranulation and activation to an allergen (dinitrophenol-conjugated human serum albumin) by measuring β-hexosaminidase release, LTB4 and IL-6 production. In addition, we assessed reactive oxygen species (ROS) generation, cell oxidative stress and toxicity as well as total and individual protein tyrosine phosphorylation (p-Tyr). We found that pre-exposure of BMMCs to AgNPs results in exacerbated allergen-mediated mast cell degranulation, LTB4 production and IL-6 release. Exposure of BMMCs to AgNPs exacerbated allergen-induced ROS generation, however, this was not associated with oxidative stress nor cell death. Finally, pre-exposure to AgNPs enhanced allergen-mediated global p-Tyr as well as individual proteins including Syk, PLCγ and LAT. Our findings indicate that pre-exposure to AgNPs exacerbates mast cell allergen-mediated phosphorylation of FcεR1-linked tyrosine kinases and ROS generation resulting in amplified early and late-phase responses. These findings suggest that exposure to AgNPs has the potential to prime mast cells to allergic immune responses, which could be of particular concern to atopic populations as the use of AgNPs in consumer and biomedical products rapidly increases.