Degranulation In RBL-2H3 Cells Research Articles

Tofacitinib suppresses mast cell degranulation and attenuates experimental allergic conjunctivitis

BackgroundAllergic conjunctivitis (AC), a common eye inflammation that affects patients’ health and quality of life, is still a therapeutic challenge for ophthalmologists. Tofacitinib, a new Janus kinase (JAK) inhibitor, has been successfully used in the treatment of several disorders. Nonetheless, its effect in AC and the potential anti-allergic mechanisms are still unclear. The objective of the current study was to explore the roles of tofacitinib in preventing AC and elucidate the potential underlying mechanisms. MethodsTofacitinib was used topically in BALB/c mice with experimental allergic conjunctivitis (EAC). Ocular allergic symptoms and biological modifications were examined. To assess the anti-allergic mechanisms of tofacitinib, RBL-2H3 cells and HUVECs were cultured in vitro. The inhibitory effects and mechanisms of tofacitinib were studied and measured by real-time quantitative PCR, ELISA, western blot analysis, and flow cytometry. ResultsTopical administration of tofacitinib reduced the clinical symptoms of OVA-induced EAC, with a substantial mitigation in inflammatory cell infiltration, histamine release, and TNF-α mRNA as well as IL-4 mRNA expression. In vitro, tofacitinib repressed the degranulation and cytokine production in RBL-2H3 cells and reduced histamine-induced vascular hyperpermeability. The underlying mechanism might involve the downregulation of phosphorylation of JAK3/STATs signaling molecules in RBL-2H3 cells and HUVECs. ConclusionsOur findings provide evidence that tofacitinib prevented EAC by targeting the JAK3/STATs pathway. We recommend the use of tofacitinib as an innovative approach for the treatment of AC.

Real-time cell analysis combined with pattern recognition to evaluate anaphylaxis of Zushima Injection

This paper was aimed to establish a new method for evaluating the anaphylactoid reaction of 15 batches of Zushima Injection from different manufacturers in vitro. Basophilic leukemia cell line RBL-2 H3 cells were cultured in vitro and Compound 48/80 was selected as positive drug. Real-time cell analysis(RTCA) system was used to detect the changes of cell index(CI) value after drug intervention. The degranulation of RBL-2 H3 cells was verified with the toluidine blue staining technology by observing the changes of cell morphology and skeleton. Clustering method was used to analyze the CI values of 15 batches of Zushima Injection on RBL-2 H3 cells. The results showed Compound 48/80(20 μg·mL~(-1)) significantly changed the cell morphology and cytoskeleton, with obvious degranulation. After adding Compound 48/80, CI value decreased rapidly within 30 minutes, then decreased slowly, suggesting that RTCA system can be used for rapid and sensitive evaluation of RBL-2 H3 cell degranulation. The results of cluster analysis showed that Zushima Injection from different manufacturers had different effects on RBL-2 H3 cells. S1-S8 and Compound 48/80 groups were grouped into one cluster, which suggesting that the sample might have potential clinical anaphylaxis. S9-S15 and the normal control group were grouped into one cluster, suggesting there was no anaphylactoid reaction in the sample. In this study, a rapid in vitro anaphylaxis evaluation technique based on RTCA system and pattern recognition method was established, which can be used for rapid in vitro evaluation of anaphylaxis for traditional Chinese medicine injection.

Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death.

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and β-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of β-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

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.

Usnic acid as calcium ionophore and mast cells stimulator

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

Pterostilbene Inhibits FcεRI Signaling through Activation of the LKB1/AMPK Pathway in Allergic Response.

In this study, the role and mechanism of pterostilbene (Pts) in mast cell degranulation in vitro and in vivo were investigated. The results showed that Pts inhibited mast cell-mediated local passive allergic reactions in mice. In addition, treatment with Pts reduced both histamine release and calcium influx in rat peritoneal mast cells and RBL-2H3 cells and reduced IgE-mediated mast cell activation. Furthermore, the mechanism underlying Pts inhibition of mast cell signaling was probed via studying the effects of Pts on liver kinase B1 (LKB1), including the use of the LKB1 activator metformin and siRNA knockdown of LKB1. The data showed that Pts reduced the release of inflammatory mediators such as tumor necrosis factor-α, interleukin-6, leukotriene C4, and prostaglandin D2 in mast cells by activating the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway. Furthermore, Pts inhibited phosphorylation of FcεRI and FcεRI-mediated degranulation in RBL-2H3 cells. These effects were attenuated after LKB1 knockdown. Taken together, Pts could inhibit FcεRI signaling through activation of the LKB1/AMPK signaling pathway in IgE-mediated mast cell activation. Thus, Pts might be an effective therapeutic agent for mast cell-mediated allergic diseases.

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.

5-HMF induces anaphylactoid reactions in vivo and in vitro

AimExcessive exposure to 5-hydroxymethylfurfural (5-HMF), which is a common impurity in various sugar-containing products, induces serious side effects. Our previous study revealed that 5-HMF exerted immune sensitizing potential when injected into rodents. In this study, we explored 5-HMF mediated anaphylactoid reactions and its underlying molecular mechanisms. MethodsWe investigated anaphylactoid reactions in Brown Norway (BN) rats and Institute of Cancer Research (ICR) mice to identify 5-HMF mediated in vivo anaphylactoid reactions. RBL-2H3 and P815 cell degranulation models were also established, and degranulation, enzyme-linked immunosorbent, filamentous actin (F-actin) microfilament staining, and western blot assays were performed in these cells. ResultsWe showed that 5-HMF induced anaphylactoid reactions by increasing blood vessel permeability in mice, and significantly elevating histamine (His) and glutathione peroxidase-1 (Gpx-1) levels in rat serum. Moreover, after incubation with 5-HMF, β-hexosaminidase (β-Hex), His, IL-4 and IL-6 levels were all significantly increased, thereby inducing cellular degranulation in RBL-2H3 and P815 cells. Finally, 5-HMF also upregulated Lyn, Syk, p38 and JNK protein phosphorylation levels. ConclusionsOur findings suggest that 5-HMF induces anaphylactoid reactions both in vivo and in vitro, therefore 5-HMF limits in sugar-containing products should receive more regulatory attention.

Antioxidant compounds of Petasites japonicus and their preventive effects in chronic diseases: a review.

Petasites japonicus (P. japonicus) is a plant of the Asteraceae family. Its roots and stems have been used for the treatment or the prophylaxis of migraine and tension headache as a traditional Chinese medicine in Japan and Korea. Sesquiterpenoids, lignans, and flavonoids are components of P. japonicus. Regarding the biological activity of P. japonicus, its anti-allergic effect has been researched extensively using IgE antigen-stimulated degranulation of RBL-2H3 cells or passive cutaneous anaphylaxis reaction in experimental animal models. The study of the antioxidant activity of P. japonicus was initiated approximately 15 years ago using in vitro assays. In addition, its in vivo effect has also been examined in animal models with induced oxidative injury. Moreover, recently, many types of antioxidant compounds have been rapidly and simultaneously identified using the liquid chromatography–mass spectrometry technique. The number of reports on the other functions of this plant, such as its neuroprotective and anti-inflammatory effects, has been increasing. In this review, I summarized the studies of functional foods derived from P. japonicus, which may provide a basis for the development of potential functional foods. Finally, I discuss the future research avenues in this field.

Comparison of the Biological Activity and Constituents in Japanese Ambers

Background/Aim: Kuji amber is an interesting natural source for drug discovery because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materials and Method: Biological activities of each extract were evaluated using growth-restoring activity of the mutant yeast strain involving Ca2+-signal transduction and inhibition activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers appeared to have the same origin. Choshi, Mizunami, and Ube ambers are valuable sources for biologically active compounds which are different from those of Kuji amber.

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.

Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity

BackgroundPalmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a “dietary food for special medical purposes” against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the “entourage” effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model.MethodsRBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and β-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -β enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively.ResultsSP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -β activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188.ConclusionsActivation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

Qi-Wei-Du-Qi-Wan and its major constituents exert an anti-asthmatic effect by inhibiting mast cell degranulation

Ethnopharmacological relevanceIn Asia, Qi-Wei-Du-Qi-Wan (QWDQW) is a traditional Chinese medicine that has been used to treat chest tightness, cough, shortness of breath, night sweats, frequent urination and asthma. QWDQW is recorded in Yi Zong Yi Ren Pian (Medical Physician's Compilation), which was written by Yang Cheng Liu during the Qing Dynasty. Aim of the studyThe traditional Chinese medicine QWDQW is composed of 7 ingredients and has been used in the treatment of asthma in Asia for hundreds of years. However, the mechanism through which QWDQW affects the immune system in the treatment of asthma is not known. Therefore, this study aimed to investigate whether QWDQW alleviates asthmatic symptoms in mice with chronic asthma induced by repeated stimulation with Dermatophagoides pteronyssinus (Der p) and to explore the underlying immune modulatory mechanism. Materials and methodsBALB/c mice were stimulated intratracheally (i.t.) with Der p (40 μl, 2.5 μg/μl) once weekly for 6 weeks. Thirty minutes prior to Der p stimulation, the mice were treated with QWDQW (0.5 g/kg and 0.17 g/kg) orally. Three days after the last stimulation, the mice were sacrificed, and infiltration of inflammatory cells, lung histological characteristics, gene expression of lung and serum total IgE were assessed. In other experiments, RBL-2H3 cells were stimulated with DNP-IgE/DNP-BSA and then treated with QWDQW, quercetin, β-carotene, luteolin or a mixture of the three chemicals (Mix13) for 30 min, and the effects of the drugs on RBL-2H3 cell degranulation after DNP stimulation were determined. ResultsQWDQW significantly reduced Der p-induced airway hyperreactivity (AHR) and decreased total serum IgE and Der p-specific IgE levels. Histopathological examination showed that QWDQW reduced inflammatory cell infiltration and sputum secretion from goblet cells in the lungs. Gene expression analysis indicated that QWDQW reduced overproduction of IL-12、IFN-γ、IL-13、IL-4、RNATES、Eotaxin and MCP-1in lung. Additionally, QWDQW and Mix13 suppressed DNP induced RBL-2H3 degranulation, and the effect was maximal when quercetin, β-carotene and luteolin were administered together. ConclusionThese results indicate that QWDQW plays a role in suppressing excessive airway reaction and in specific immune modulation in a mouse model of chronic asthma and that QWDQW suppresses mast cell degranulation at defined doses of quercetin, β-carotene and luteolin.

Docosahexaenoyl ethanolamide mitigates IgE-mediated allergic reactions by inhibiting mast cell degranulation and regulating allergy-related immune cells

Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid mainly found in fish oil. Although several studies have suggested that it can alleviate allergy symptoms, its mechanism of action remains to be elucidated. In the present study, we found that docosahexaenoyl ethanolamide (DHEA), a metabolite of DHA produced in the human body, exerts the anti-allergic activity in vitro and in vivo. DHEA suppressed degranulation of rat basophilic leukemia RBL-2H3 cells and bone marrow-derived mast cells in a dose-dependent manner without cytotoxicity. This occurred due to a decrease in Ca2+ influx, which is critical for mast cell degranulation. DHEA also suppressed IgE-mediated passive cutaneous anaphylaxis reaction in mice. In addition, DHEA was demonstrated to lessen an allergic symptom in a mouse model of pollinosis and to alter the production of IgE and cytokines secreted by splenocytes collected from the pollinosis mice. Taken together, this study indicates that DHEA is a promising anti-allergic agent as it inhibits mast cell degranulation and modulates other immune cells.

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.

Study on safflower yellow for injection based on cell degranulation and acute anaphylactoid model

This paper was aimed to establish screening methods of anaphylactoid reaction caused by safflower yellow for injection based on RBL-2 H3 cell degranulation model and mice model for acute anaphylactoid reaction,and evaluate the hypersensitivity caused by safflower yellow for injection from different batches. An in vitro cell model was used to keep the cells stimulated for an hour with different batches of safflower yellow for injection as the drug group,serum-free MEM medium as negative control group and 30 mg·L-1 C48/80 as positive control group respectively. The supernatant was then absorbed,and neutral red staining technique was used to detect the effect of safflower yellow injection on the degranulation of RBL-2 H3 cells with the positive cell rate of degranulation as the indicator.An in vivo model was established to validate the experimental results,and mice model for acute anaphylactoid reaction and ELISA method were adopted to detect the plasma histamine content,and screen the hypersensitivity caused by safflower yellow for injection at the animal level by using plasma histamine content as a test index. The results of the neutral red staining experiments showed that the positive control C48/80 could cause cell degranulation,and most of the cells were deeply stained. There was significant difference in positive cell rate between different batches of safflower yellow and positive control group. In the mice model for acute anaphylactoid reaction,it was found that the positive control C48/80 significantly increased the histamine content in the plasma of mice,while the safflower yellow in each batch did not cause a significant increase in plasma histamine( P<0. 000 1). The mechanism of anaphylactoid reaction is relatively complicated. This study was mainly based on the release of histamine and other active substances by degranulation of mast cells. No significant degranulation reaction of RBL-2 H3 cells induced by safflower yellow for injection was detected,nor was the plasma histamine level significantly increased in mice from the in vitro and in vivo aspects.

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells.

Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE488) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.

The composition and anti-inflammatory effect of polysaccharides from the red alga Chondrus verrucosus

Polysaccharides prepared from the red alga Chondrus verrucosus (Rhodophyta, Gigartinales) were subjected to anion-exchange column chromatography, and the chemical characteristics of the fractions (designated CV1, CV2 and CV3 in the order of elution) were examined based on carbohydrate and sulfate contents and monosaccharide composition. Furthermore, the anti-inflammatory effect of the fractions on the degranulation in RBL-2H3 cells stimulated by A23187 was investigated. The results showed that the major monosaccharide component was galactose. The CV1 and CV2 fractions showed higher anti-inflammatory activity against RBL-2H3 cells than the CV3 fraction. The difference in activity may be related to the sulfate contents, namely, the contents of CV1, CV2 and CV3 fractions were 25.3 ± 3.3%, 28.1 ± 1.1%, and 7.4 ± 1.8%, respectively.

Mast cell stabilizing effect of a geranyl acetophenone in dengue virus infection using in vitro model of DENV3-induced RBL-2H3 cells.

Mast cells (MCs), a type of immune effector cell, have recently become recognized for their ability to cause vascular leakage during dengue virus (DENV) infection. Although MC stabilizers have been reported to attenuate DENV induced infection in animal studies, there are limited in vitro studies on the use of MC stabilizers against DENV induced MC degranulation. 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA) has been reported to be a potential MC stabilizer by inhibiting IgE-mediated MC activation in both cellular and animal models. The present study aims to establish an in vitro model of DENV3-induced RBL-2H3 cells using ketotifen fumarate as a control drug, as well as to determine the effect of tHGA on the release of MC mediators upon DENV infection. Our results demonstrated that the optimal multiplicities of infection (MOI) were 0.4 × 10−2 and 0.8 × 10−2 focus forming units (FFU)/cell. Ketotifen fumarate was proven to attenuate DENV3-induced RBL-2H3 cells degranulation in this in vitro model. In contrast, tHGA was unable to attenuate the release of both β-hexosaminidase and tumor necrosis factor (TNF)-α. Nonetheless, our study has successfully established an in vitro model of DENV3-induced RBL-2H3 cells, which might be useful for the screening of potential MC stabilizers for anti-dengue therapies.

Inhibitory Effects of Phenylpropanoid Derivatives from Oenanthe javanica on Antigen-Stimulated Degranulation in RBL-2H3 Cells.

Two diacyldaucic acids (1 and 2), an α,β-unsaturated γ-lactone-type lignan (3) and its derivatives (4-6), and 12 known compounds were isolated from a traditional East Asian vegetable, Oenanthe javanica. The absolute configuration of 1 was validated by obtaining (+)-osbeckic acid through acid hydrolysis. The absolute configurations of 3-5 were determined by comparing their experimental and computed ECD data. The conclusion was supported by applying the phenylglycine methyl ester method to 3. Compound 6 was obtained as an interconverting mixture of isomers in a 3:1 trans- cis ratio. Several water-soluble components (1, 3, and 6) showed concentration-dependent inhibitory effects on antigen-stimulated degranulation in RBL-2H3 cells without producing any direct cytotoxicity against RBL-2H3 or HeLa cells.