Anti-anaphylactic potential of benzoylpaeoniflorin through inhibiting HDC and MAPKs from Paeonia lactiflora / 中国天然药物

Guided by cell-based anti-anaphylactic assay, eighteen cage-like monoterpenoid glycosides (1-18) were obtained from the bioactive fraction of P. lactiflora extract. Among these, compounds 1, 5, 6, 11, 12, 15, and 17 significantly reduced the release rate of β-HEX and HIS without or with less cytotoxicity. Furthermore, the most potent inhibitor benzoylpaeoniflorin (5) was selected as the prioritized compound for the study of action of mechanism, and its anti-anaphylactic activity was medicated by dual-inhibiting HDC and MAPK signal pathway. Moreover, molecular docking simulation explained that benzoylpaeoniflorin (5) blocked the conversion of L-histidine to HIS by occupying the HDC active site. Finally, in vivo on PCA using BALB/c mice, benzoylpaeoniflorin (5) suppressed the IgE-mediated PCA reaction in antigen-challenged mice. These findings indicated that cage-like monoterpenoid glycosides, especially benzoylpaeoniflorin (5), mainly contribute to the anti-anaphylactic activity of P. lactiflora by dual-inhibiting HDC and MAPK signal pathway. Therefore, benzoylpaeoniflorin (5) may be considered as a novel drug candidate for the treatment of anaphylactic diseases.

Safety study and compositional analysis of polyoxyethylene 35 castor oil by UPCC-Q-TOF-MS / 药学学报

A UPCC-Q-TOF-MS method was established to analyze the components of polyoxyethylene 35 castor oil. The separation was performed at 50 ℃ on a Waters Acquity UPCC system by an Torus Diol column (3.0 mm × 100 mm, 1.7 μm) with gradient elution of CO2 and methanol - acetonitrile (50∶50); the flow rate was 1.0 mL·min-1, the back pressure was 2 000 psi, and methanol containing 2.5 mmol·L-1 ammonium formate was used as ionization reagent, whose flow rate was 0.2 mL·min-1. Positive ion electrospray ionization (ESI) mode and MSE technology were used. The qualitative analyses were carried out by using precise mass information of the parent and product ions and a PCA model was established by UPCC-Q-TOF-MS. L-02 cells and RBL-2H3 cells were used to study the cytotoxicity and histamine release of CrEL samples in vitro. A total of 13 kinds of CrEL components were obtained and their structures were identified by UPCC-Q-TOF-MS, with 255 compounds in total. The percentage content of 13 types of components was calculated by the normalization method. The content of polyoxyethylene glycerol tri-ricinoleate (PGTri-ricinoleate) in all samples was 0.36% - 2.80% and the main components were polyethylene glycol, polyethylene glycerol and polyoxyethylene glycerol mono-ricinoleate. All samples have different degrees of cytotoxicity and histamine release, which is negatively correlated with the content of PGTri-ricinoleate and positively correlated with the content of polyoxyethylene glycol fatty acid esters. The UPCC-Q-TOF-MS method is simple and rapid, has strong separation ability and high accuracy. It is suitable for the analysis of CrEL components. It is suggested that the fatty acid composition should be included in the monograph of CrEL for injection to increase the content of PGTri-ricinoleate and decrease the content of polyoxyethylene glycol fatty acid esters, so as to improve the product safety.

Metabolomic study of Gardenia jasminoside var. radicans and geniposide on degranulation model of RBL-2H3 cells / 中草药

Objective: To study the intervention effect of Gardenia jasminoside var. radicans and its main effective component of geniposide on the degranulation model of RBL-2H3 cells based on metabolomics. Methods: The changed metabolite profile of RBL-2H3 cells was detected by UPLC-QTOF-MS; PCA (principal component analysis) and OPLS-DA (orthogonal partial least squares discriminant analysis) in SIMCA software were used to select the potential biomarkers. Meanwhile, the clustering and heat map analysis for those potential biomarker levels were carried out by MEV software. Result: A total of 54 and 46 relevant biomarkers of G. jasminoside var. radicans and geniposide were selected, of which 31 biomarkers enriched in five disturbed metabolite pathways, including glycine, aspartic acid and glutamate metabolism, glutathione metabolism, histamine metabolism, energy metabolism, and nicotinamide metabolism pathways. Conclusion: G. jasminoside var. radicans and geniposide exerts the inhibitory effect on the degranulation model of RBL-2H3 cells by regulating histamine metabolism, oxidative stress and energy metabolism, and geniposide was one of the main efficacious substance basis of G. jasminoside var. radicans.

Suppressive Effect of 4-Hydroxy-2-(4-Hydroxyphenethyl) Isoindoline-1,3-Dione on Ovalbumin-Induced Allergic Asthma

4-Hydroxy-2-(4-hydroxyphenethyl)isoindoline-1,3-dione (PD1) is a synthetic phthalimide derivative of a marine compound. PD1 has peroxisome proliferator-activated receptor (PPAR) γ agonistic and anti-inflammatory effects. This study aimed to investigate the effect of PD1 on allergic asthma using rat basophilic leukemia (RBL)-2H3 mast cells and an ovalbumin (OVA)-induced asthma mouse model. In vitro, PD1 suppressed β-hexosaminidase activity in RBL-2H3 cells. In the OVA-induced allergic asthma mouse model, increased inflammatory cells and elevated Th2 and Th1 cytokine levels were observed in bronchoalveolar lavage fluid (BALF) and lung tissue. PD1 administration decreased the numbers of inflammatory cells, especially eosinophils, and reduced the mRNA and protein levels of the Th2 cytokines including interleukin (IL)-4 and IL-13, in BALF and lung tissue. The severity of inflammation and mucin secretion in the lungs of PD1-treated mice was also less. These findings indicate that PD1 could be a potential compound for anti-allergic therapy.

Effects of carboxyamidotriazole on the activation and degranulation of RBL-2H3 cells / 基础医学与临床

Objective To explore the anti-infection mechanism of carboxyamidotriazole (CAI) through studying the effects of CAI on the proliferation, apoptosis and degranulation of RBL-2H3 mass cells.Methods Compound 48/80 (C48/80) was used to induce the model of activation and degranulation in RBL-2H3 cells.The morphological change of cell degranulation was observed by neutral red staining.The release levels of histamine and β-hexosaminidase were measured by ELISA method and chromogenic assay, respectively.The cell activity was determined by CCK-8 method.And cell apoptosis was detected by Hoechst 33342 fluorescent staining.Results Compared with the control group, 10, 20, 40 μmol/L CAI inhibited C48/80-induced degranulation of RBL-2H3 cells in different degrees.CAI (20, 40 μmol/L) reduced the histamine release (P<0.01), and CAI (40 μmol/L) decreased the β-hexosaminidase release (P<0.01).In addition, the viability and apoptosis of RBL-2H3 cells were not affected at the concentrations of CAI used.Conclusions CAI can effectively inhibit the activation and degranulation of RBL-2H3 mast cells, and this effect is not through cytotoxicity.The anti-infection effect of CAI may partially due to the down-regulation of mast cell activity.

A novel arctigenin-containing latex glove prevents latex allergy by inhibiting type I/IV allergic reactions / 中国天然药物

The present study aimed at developing a natural compound with anti-allergic effect and stability under latex glove manufacturing conditions and investigating whether its anti-allergic effect is maintained after its addition into the latex. The effects of nine natural compounds on growth of the RBL-2H3 cells and mouse primary spleen lymphocytes were determined using MTT assay. The compounds included glycyrrhizin, osthole, tetrandrine, tea polyphenol, catechin, arctigenin, oleanolic acid, baicalin and oxymatrine. An ELISA assay was used for the in vitro anti-type I/IV allergy screening; in this process β-hexosaminidase, histamine, and IL-4 released from RBL-2H3 cell lines and IFN-γ and IL-2 released from mouse primary spleen lymphocytes were taken as screening indices. The physical stability of eight natural compounds and the dissolubility of arctigenin, selected based on the in vitro pharnacodynamaic screening and the stability evaluation, were detected by HPLC. The in vivo pharmacodynamic confirmation of arctigenin and final latex product was evaluated with a passive cutaneous anaphylaxis (PCA) model and an allergen-specific skin response model. Nine natural compounds showed minor growth inhibition on RBL-2H3 cells and mouse primary spleen lymphocytes. Baicalin and arctigenin had the best anti-type I and IV allergic effects among the natural compounds based on the in vitro pharmacodynamic screening. Arctigenin and catechin had the best physical stability under different manufacturing conditions. Arctigenin was the selected for further evaluation and proven to have anti-type I and IV allergic effects in vivo in a dose-dependent manner. The final product of the arctigenin-containing latex glove had anti-type I and IV allergic effects in vivo which were mainly attributed to arctigenin as proved from the dissolubility results. Arctigenin showed anti-type I and IV allergic effects in vitro and in vivo, with a good stability under latex glove manufacturing conditions, and a persistent anti-allergic effect after being added into the latex to prevent latex allergy.

Hemolytic assay for Huangqi Injection / 中草药·英文版

Objective: Huangqi Injection is a preparation with an extract of Astragali Radix which has a long history of being used as a tonic to strengthen the body's immunity. Anaphylaxis and hemolysis are two main adverse drug reaction (ADR) of injections. Our study was aimed to establish an approach for the (ADR) prediagnosis of Huangqi Injection. Methods: An in vitro model for anaphylactoid assay of Huangqi Injection based on the release rate of histamine and β-hexosaminidase of RBL-2H3 cells induced by injections and a colorimetric method based on the detection of hemoglobin resulted in the erythrocyte hemolysis for prediagnostic assaying the hemolytic ADR of injections were established. Results: Both histamine and β-hexosaminidase are the anaphylactiod mediators, but β-hexosaminidase release induced by Huangqi Injection could not be determined by spectrophotometry due to the interference of the injection itself. In addition, normal hemolysis and abnormal hemolysis were discovered during the experiment. The fingerprints and tannins in different batches of injections showed obvious differences, indicating that the content of tannins was related to abnormal hemolysis and higher histamine-secreting from RBL-2H3 cells. Conclusion: The results indicate that the hemolytic assaying method is not only suitable for prediagnostic assaying of hemolytic ADR of herbal medicine injection, but also partly reflects the anaphylaxis of herbal injections, and tannins may be the major factors causing abnormal hemolysis.

The in vitro and in vivo models induced by shrimp tropomyosin and monoclonal tropomyosin-specific murine IgE antibody / 中国药理学通报

Aim To develop the in vitro and in vivo models induced by shrimp tropomyosin( ST) and mono-clonal tropomyosin-specific murine IgE antibody ( anti-ST-IgE mAb) . Methods ST was purified from Metap-enaeusensis by an isoelectric precipitation method. The anti-ST-IgE mAb was obtained from hybridomas. After RBL-2 H3 cells were sensitized with anti-ST-IgE mAb and challenged with ST,β-hexosaminidase release was determined. Passive systemic anaphylaxis ( PSA ) was induced in mice and the rectal temperature was recor-ded after ST challenge within 30 min by a thermal probe. Results A significant increase ofβ-hexosamin-idase was observed in sensitized cells after ST chal-lenge. The average temperature drop after ST challenge was 1. 44℃ in PSA mice within 30 min. Conclusion The in vitro and in vivo models induced by ST and anti-ST-IgE mAb are established as an improvement of pres-ent models of type Ⅰ allergy.

Application of RBL-2H3 cells on allergenicity assessment of Qing kailing and Tanreqing Injections / 中草药

Objective: To explore the allergenicity of two kinds of Chinese materia medica (CMM) injections, Qingkailing (QKL) and Tanreqing (TRQ) Injections, with serum antibody sensitized RBL-2H3 cells. Methods: The antiserum was prepared by sc injection of allergen composed of ovalbumin (OVA), QKL, or TRQ combined with aluminum hydroxide adjuvant respectively in Wistar rats. The total IgE level in serum antibody was determined by radioimmunoassay. The RBL-2H3 cells were sensitized with serum antibodies, then stimulated by OVA, QKL, or TRQ after 48 h. The release rate of β-hexosaminidase in the supernatant was determined after the degranulation of sensitized RBL-2H3 cells. Passive cutaneous anaphylaxis (PCA) test was performed with rats, and the positive reaction rate with blue plaque in animal skin was observed. Results: Compared with the control group, the total IgE level in serum antibody was increased significantly in OVA, TRQ, and QKL groups (P < 0.05). The degranulation test revealed that the release rate of β-hexosaminidase was significantly increased in the supernatant when the cells were incubated with the antiserum and then stimulated with OVA, QKL, and TRQ. Compared with the control group, the largest relative times of release were 3.7, 1.53, and 1.98, respectively. The results of PCA test showed that the highest percentage rates of positive reaction of blue plaque were 100%, 100%, and 86% respectively. The results of RBL-2H3 cell test and PCA test have good consistency. Conclusion: The serum antibody sensitized RBL-2H3 cell model can be used for screening or assessing allergenicity of CMM injection.

Construction and identification of recombinant RBL-2 H3 cells transfected with hFcεRIα / 中国药理学通报

Aim To construct the stable hFcεRIα/RBL-2H3 cell line expressing human FcεRIα( hFcεRIα) . Methods The human FcεRIα gene was obtained by RT-PCR and cloned into the eukaryotic expression vector pCI-neo. Then, the pCI-neo-hFcεRIα vector was transfected into RBL-2H3 cells by lipo-somes, and the transfected cells were screened through G418 fil-tration subsequently. Finally, RT-PCR, Western blot and immu-nofluorescence assay were used to determine the result of trans-fection. Results According to the optimized transfection param-eters, the transfection efficiency reached 75. 38%. The results of Western blot, immunofluorescence and RT-PCR showed that hFcεRIα could be expressed in RBL-2H3 cells successfully. Conclusion HFcεRIα/RBL-2H3 cells were successfully con-structed,which will be the experimental basis for further study on the mechanism of IgE/FcεRI and drugs for allergy diseases.

Studies on Anaphylactoid Reactions Induced by Traditional Chinese Medicine Injections of Qingkailing and Xuesaitong / 中国中医药信息杂志

Objective To study anaphylactoid reactions induced by traditional Chinese medicine injections (TCMI) of Qingkailing (QKL) and Xuesaitong (XST) with RBL-2H3 cells;To provide some reference for improving the screening system of TCMI induced anaphylactoid reactions.Methods The IC50 induced by QKL and XST injections was determined by MTT assay. RBL-2H3 cells were stimulated with TCMI at different concentrations or with C48/80 or culture medium. Thirty minutes later, the supernatant was collected to determine the release of histamine andβ-hexosaminidase. The cell degranulation rate and the ultrastructure changes were observed. The ICR mice were given single injection of TCMI containing Evans Blue through tail vein. The number of the animal with blue ear, the total number of blue ears and the quantity of Evans Blue of extravasation were determined 30 minutes later.Results The IC50 of both QKL injection and XST injection was 12.5μL/mL. These two injections promoted RBL-2H3 cells to release histamine andβ-hexosaminidase at higher concentrations (P<0.05,P<0.01) in a dose dependent manner. Cell morphology showed a decrease of villous on the cell surface and an increase of the internal vacuolated structure. Both injections caused the blue ears of all animal with a rate of 100%. The quantity of Evans Blue of the extravasation was significantly increased (P<0.01). The results in vitro study were in close agreement with that in vivo.Conclusion Both QKL injection and XST injection may potentially cause anaphylactoid reactions. The RBL-2H3 cell model may be valuable to evaluate the anaphylactoid reactions induced by TCMI.

Replacing solubilizer with lipid microspheres eliminates the severe adverse reaction of vitamin K1 injection / 中国药学杂志

OBJECTIVE: To investigate whether substituting the solubilizer with lipid microspheres in vitamin K1 injection can eliminate the anaphylactoid reaction.

"Influences of ""Antiphlogistic No.1"" on the degranultion of Activated RBL-2H3 cells" / 国际中医中药杂志

Objective To investigate the direct effect ofAntiphlogistic No.1 on the degranultion of RBL-2H3 mast cells stimulated by anti-DNP IgE and DNP-BSA complex.Methods The effect of Antiphlogistic No.1 on stabilization of RBL-2H3 cell membrane was assessed by degranultion inhibition rate.β-hexosaminidase and IL-4 released from RBL-2H3 cells were detected by PNAG colorimetric assay and ELISA.Results β-hexosaminidase and cytokine IL-4 production releases from RBL-2H3 cells was reduced after they had been treated with Antiphlogistic No.1.The inhibition rate of β-hexosaminidase release was 42.47％ at the concentration of 8 μg/ml (P＜0.01) and dose dependently increased to 52.40％,68.26％ and 72.15％ respectively when drug concentration up to 80 μg/ml,800 μg/ml,8000 μg/ml,while inhibition rate of IL-4 generation were 13.87％,23.27％,31.95％,39.99％ (P＜0.01).Antiphlogistic No.1 maximum inhibition rate of β-hexosaminidase release is 84.48％ of dexamethason maximum inhibition rate while Antiphlogistic No.l maximum inhibition rate of IL-4 is close to dexamethason maximum inhibition rate.Conclusion Antiphlogistic No.1 could stabilize the cellular membrane of RBL-2H3 and provide protection against Ⅰ type allergic response.

Suppressive effects of Schizandra chinensis Baillon water extract on allergy-related cytokine generation and degranulation in IgE-antigen complex-stimulated RBL-2H3 cells

Schizandra chinensis Baillon is a traditional folk medicine plant that is used to treat and prevent several inflammatory diseases and cancer in Korea, but the underlying mechanisms involved in its anti-allergic activity are not fully understood. This study was designed to investigate mechanisms of anti-allergic activity of a Schizandra chinensis Baillon water extract (SCWE) in immunoglobulin E (IgE)-antigen complex-stimulated RBL2H3 cells and to assess whether gastric and intestinal digestion affects the anti-allergic properties of SCWE. Oxidative stress is an important consequence of the allergic inflammatory response. The antioxidant activities of SCWE increased in a concentration-dependent manner. RBL-2H3 cells were sensitized with monoclonal anti-dinitrophenol (DNP) specific IgE, treated with SCWE, and challenged with the antigen DNP-human serum albumin. SCWE inhibited beta-hexosaminidase release and expression of interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein in IgE-antigen complex-stimulated RBL2H3 cells. We found that digested SCWE fully maintained its antioxidant activity and anti-allergic activity against the IgE-antigen complex-induced activation of RBL-2H3 cells. SCWE may be useful for preventing allergic diseases, such as asthma. Thus, SCWE could be used as a natural functional ingredient for allergic diseases in the food and/or pharmaceutical industries.

Anti-Oxidative and Anti-Inflammatory Effect of Water Extract from Perillae semen in RBL-2H3 Cells

The present study was to investigate anti-oxidative and anti-inflammatory activity of Perillae semen in RBL-2H3 basophilic leukemia cells. Inhibitory effect of Perillae semen onto free radical generation was determined by measuring DPPH and hydroxyl radical scavenging activities in vitro. Anti-inflammatory actions of Perillae semen extracts (100, 250, 500 microgram/mL) were assessed by testing their effects on the degranulation of mast cells. For this, beta-hexosaminidase released from RBL-2H3 cells was used and proinflammatory cytokines were measured by an ELISA kit. Our results indicated that Perillae semen water extracts effectively inhibited free radical generation. At the concentration of 500 microgram/mL of water extract, the degranulation of RBL-2H3 cells were inhibited by 42.1%. The IgE-antigen complex increased the accumulation of IL-4 and TNF-alpha secretion in RBL-2H3 cells and treatments with 250 and 500 microgram/mL of Perillae semen extracts suppressed the IgE induced secretion of IL-4 and TNF-alpha protein by 20.5, 26.9% and 14.5, 16.5% respectively. We observed that Perillae semen water extract reduced beta-hexosaminidase, IL-4, and TNF-alpha secretion in RBL-2H3 cells. These results provide that Perillae semen may be beneficial in the treatment of allergic inflammatory disease.

A Synthetic Chenodeoxycholic Acid Derivative, HS-1200-induced Apoptosis of RBL-2H3 Cells / 대한해부학회지

Bile acids and synthetic bile acid derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. Although synthetic chenodeoxycholic acid (CDCA) derivatives have been demonstrated to induce apoptosis of various cancer cells, there is no report on their effect on RBL-2H3 basophilic leukemia cell line to date. Therefore, this study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in RBL-2H3 cells treated with a synthetic CDCA derivative, HS-1200. The viability and the growth inhibition of RBL-2H3 cells were assessed by MTT assay and clonogenic assay respectively. The Hoechst staining and DNA electrophoresis were conducted to observe RBL-2H3 cells undergoing apoptosis. RBL-2H3 cells were treated with HS-1200, and Western blotting, immunocytochemistry, confocal microscopy, DNA hypoploidy assay, MMP activity and proteasome activity were performed. HS-1200 treatment of RBL-2H3 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Furthermore, HS-1200 treatment result in the alteration of G1 cell cycle-related proteins. And tested RBL-2H3 cells showed several lines of apoptotic manifestation.We presented data indicating that HS-1200 induces apoptois via the proteasome, mitochondria and caspase pathway, and induces the alteration of the G1 cell cycle-related proteins in RBL-2H3 cells. Therefore our data provide the possibility that HS-1200 could be as a novel therapeutic strategy in the allergy treatment.

Fluoxetine-induced Changes on Activity of Tryptophan Hydroxylase at RBL-2H3 Cells / 대한정신약물학회지

OBJECTIVES: The aim of the present study is to explore the effect of fluoxetine on transcription, translation and activity of tryptophan hydroxylase (TPH), and intracellular level of serotonin. METHODS: The expression level of the TPH mRNA and the protein, the TPH enzyme activity, and the intracellular level of serotonin were explored at the fluoxetine-treated RBL-2H3 cells. Real-time RT-PCR and immunoblotting analysis confirmed changes in the expression of TPH mRNA and protein. The activity of TPH was measured using [3H]tryptophan. The intracellular level of serotonin was measured by HPLC. RESULTS: The TPH activity was gradually increased on time from 24hr to 72hr. The real-time RT-PCR also revealed that the TPH mRNA was increased at 12, 24 and 72hr in the fluoxetine-treated RBL-2H3 cells. The immunoblotting analysis also revealed that the TPH protein was decreased at 72hr in the fluoxetine-treated RBL-2H3 cells. The intracellular level of serotonin was increased at 48hr after treatment of fluoxetine. CONCLUSION: Fluoxetine induced the increases of the TPH mRNA, the TPH enzyme activity and intracellular level of serotonin, and the decrease of the TPH protein expression at the RBL- 2H3 cells.

Study of diagnose guinea pigs anaphylaxis shock with RBL-2H3 cells / 中国免疫学杂志

Objective:In order to study of diagnose anaphylactic shock in forensic field,a new in vitro testing sysytem were established.Methods:The RBL 2H3 were cultured and sensitized,the release of histamine and stained for Annexin V were testing by fluorescent spectrometer and flow cytometry respectively.Results:The degree of RBL 2H3 degranulation after sensitived serum treated was higher than that after control serum treated,and the degranulation degree increased dose dependently.Conclusion:RBL 2H3 are helpful to diagnose anaphylactic shock,and the release of histamine and the positive staining for Annexin V may be the ideal indices.

Fas Ligand gene transfection induces rat mast cells apoptosis / 中国免疫学杂志

Objective:To show whether the Fas Ligand gene induces mast cells apoptosis.Methods:RT-PCR was used to amplify the gene of rat Fas ligand extracelluar domain and transmembrane domain and cloned it into eukaryotic expression plasmid pcDNA3.1.Transcent transfect RBL-2H3,the expression of Fas ligand RBL-2H3 was detected by RT-PCR、Western blot.The Annexin V FCM was used to detect the RBL-2H3 apoptosis after the transfection of Fas Ligand.Results:It is successful to obtain the gene of rat Fas Ligand extracellular domain and transmembrane segment,cloning it into pcDNA3.1,FasL was expressed on the surface of RBL-2H3 and it's supernatant after the transfection of pcDNA3.1/FasL.The cell start to be apoptosis.Conclusion:Our study reveals that Fas Ligand gene transfection in RBL-2H3 can effectively induced apoptosis.It is a promising strategy for Fas Ligand to be used in the therapy of allergic disease. [