Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections

Tang, Jiaming; Liu, Jiong; Wu, Wen‐Bin

doi:10.4236/cm.2012.34029

Cited by 10 publications

(7 citation statements)

References 3 publications

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“…Other researchers have previously reported a lack of certain tryptases in RBL‐2H3 cells. Similar to our Figure results, two other studies also observed a lack of tryptase via BAPNA assay in supernatants of degranulated RBL‐2H3 cells, even while β‐hex release was readily measured in the same supernatants (Peng et al, ; Tang, Lie, & Wu, ). Mass spectrometry analyses of cell supernatants from degranulated RBL‐2H3s also found no tryptase proteins (Sadroddiny et al, ; Sadroddiny, Moir, & Helm, ).…”

Section: Discussionsupporting

confidence: 91%

Searching for tryptase in the RBL‐2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish

Shim

Kennedy

Weatherly

et al. 2018

J of Applied Toxicology

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Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes β‐hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N‐α‐benzoyl‐dl‐Arg‐p‐nitroanilide (BAPNA). An extensively used in vitro mast cell model for studying toxicant mechanisms is the RBL‐2H3 cell line. However, instead of tryptase, granule contents such as β‐hexosaminidase have usually been employed as RBL‐2H3 degranulation markers. To align RBL‐2H3 cell toxicological studies to in vivo mast cell studies using zebrafish, we aimed to develop an RBL‐2H3 tryptase assay. Unexpectedly, we discovered that tryptase release from RBL‐2H3 cells is not detectable, using BAPNA substrate, despite optimized assay that can detect as little as 1 ng tryptase. Additional studies performed with another substrate, tosyl‐Gly‐Pro‐Lys‐pNA, and with an enzyme‐linked immunosorbent assay, revealed a lack of tryptase protein released from stimulated RBL‐2H3 cells. Furthermore, none of the eight rat tryptase genes (Tpsb2, Tpsab1, Tpsg1, Prss34, Gzmk, Gzma, Prss29, Prss41) is expressed in RBL‐2H3 cells, even though all are found in RBL‐2H3 genomic DNA and even though β‐hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize β‐hexosaminidase or another reliable marker for RBL‐2H3 degranulation studies, not tryptase. Comparative toxicity testing in RBL‐2H3 cells in vitro and in zebrafish mast cells in vivo will require use of a degranulation reporter different from tryptase.

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Section: Discussionsupporting

confidence: 91%

Searching for tryptase in the RBL‐2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish

Shim

Kennedy

Weatherly

et al. 2018

J of Applied Toxicology

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“…β-Hexosaminidase release from mast cells was examined as described previously with some modifications [9]. …”

Section: Methodsmentioning

confidence: 99%

“…β-Hexosaminidase is an enzyme contained in the cytoplasmic granules of mast cells and the level of this enzyme released into the supernatant provides an indication of the degree of degranulation when mast cell is activated. β-Hexosaminidase release from mast cells was examined as described previously with some modifications [ 9 ].…”

Section: Methodsmentioning

confidence: 99%

“…However, along with the wide use of red ginseng, adverse drug reactions occur frequently, especially anaphylactoid reactions (ARs). Several red ginseng-containing TCMIs [ 9 ] or ginsenoside-containing TCMIs [ 10 ] have been shown to cause ARs that may be related to components in red ginseng. Moreover, several cases of ARs induced by oral administration of ginseng or inhalation of ginseng dust have also been recently reported [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

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Allergens in red ginseng extract induce the release of mediators associated with anaphylactoid reactions

et al. 2017

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BackgroundAnaphylactoid reactions induced by preparations containing red ginseng have been reported. The aim of this study is to evaluate the allergenicity and screen potential allergens in red ginseng extract thoroughly.MethodsRed ginseng extract (RGE) and different fractions of RGE were prepared and evaluated by measuring the degranulation and viability of rat basophilic leukemia 2H3 (RBL-2H3) cells. Potential allergens were screened by RBL-2H3 cell extraction and allergenicity verified in RBL-2H3 cells, mouse peritoneal mast cells, Laboratory of Allergic Disease 2 (LAD2) human mast cells and mice, respectively.Results80% ethanol extract of red ginseng extract induced mast cell degranulation with less cytotoxicity, but 40% ethanol extract could not. Ginsenoside Rd and 20(S)-Rg3 could induce a significant increase in β-hexosaminidase release, histamine release and translocation of phosphatidylserine in RBL-2H3 cells. Ginsenoside Rd and 20(S)-Rg3 also increased β-hexosaminidase release and the intracellular Ca2+ concentration in mouse peritoneal mast cells and LAD2 cells. In addition, histamine levels in serum of mice were elevated dose-dependently.ConclusionsGinsenoside Rd and 20(S)-Rg3 are potential allergens that induce the release of mediators associated with anaphylactoid reactions. Our study could guide optimization of methods associated with Rd/20(S)-Rg3-containing preparations and establishment of quality standards for safe application of Traditional Chinese Medicines.

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“…The extract revealed to be composed by several organic and fatty acids. These compounds could affect the results of degranulation since degranulation phenomenon is influenced by pH [19]. However, as the pH range of the medium used in the assays was buffered to pH 6.8–7.2, the amounts of acids in the extracts were not sufficient to exceed the buffering capacity of the culture medium and so degranulation was not affected.…”

Section: Resultsmentioning

confidence: 99%

Effects of Echium plantagineum L. Bee Pollen on Basophil Degranulation: Relationship with Metabolic Profile

et al. 2014

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This study aimed to evaluate the anti-allergic potential of Echium plantagineum L. bee pollen and to characterize its primary metabolites. The activity of E. plantagineum hydromethanolic extract, devoid of alkaloids, was tested against β-hexosaminidase release in rat basophilic leukemic cells (RBL-2H3). Two different stimuli were used: calcium ionophore A23187 and IgE/antigen. Lipoxygenase inhibitory activity was evaluated in a cell-free system using soybean lipoxygenase. Additionally, the extract was analysed by HPLC-UV for organic acids and by GC-IT/MS for fatty acids. In RBL-2H3 cells stimulated either with calcium ionophore or IgE/antigen, the hydromethanolic extract significantly decreased β-hexosaminidase release until the concentration of 2.08 mg/mL, without compromising cellular viability. No effect was found on lipoxygenase. Concerning extract composition, eight organic acids and five fatty acids were determined for the first time. Malonic acid (80%) and α-linolenic acid (27%) were the main compounds in each class. Overall, this study shows promising results, substantiating for the first time the utility of intake of E. plantagineum bee pollen to prevent allergy and ameliorate allergy symptoms, although a potentiation of an allergic response can occur, depending on the dose used.

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Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections

Cited by 10 publications

References 3 publications

Searching for tryptase in the RBL‐2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish

Searching for tryptase in the RBL‐2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish

Allergens in red ginseng extract induce the release of mediators associated with anaphylactoid reactions

Effects of Echium plantagineum L. Bee Pollen on Basophil Degranulation: Relationship with Metabolic Profile

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