RBL-2H3和P815细胞用于建立体外肥大细胞脱颗粒模型的比较研究(英文)

目的:通过比较两种肥大细胞模型RBL-2H3和P815细胞激活后脱颗粒反应的差异,寻找早期、稳定、敏感的肥大细胞脱颗粒体外检测模型,为进一步建立基于细胞水平的过敏原和抗过敏药物的体外筛选模型提供一定的参考。方法:10μg.mL-1C48/80激活细胞15-60min,比色法检测组胺、β-氨基己糖苷酶及类胰蛋白酶等过敏介质的释放,中性红染色法进行形态学观察,流式细胞法检测AnnexinV阳性细胞率。结果:C48/80刺激P815、RBL-2H3细胞15-60min后组胺释放率均明显增加,并且相同刺激条件下,两种细胞模型的组胺释放率基本相当。C48/80刺激P815细胞15min以上,β-氨基己糖苷酶释放率、类胰蛋白酶释放率、Annexin V阳性细胞率和中性红染色脱颗粒细胞率均明显增加;但相同浓度的C48/80需刺激RBL-2H3细胞30-45min以上,上述指标才出现显著增加。在相同的刺激条件下,P815细胞过敏介质释放率、AnnexinV阳性率和脱颗粒率均高于RBL-2H3细胞。结论:同RBL-2H3相比,在相同刺激条件下,P815细胞活化后脱颗粒时间出现更早、程度更高。提示除RBL-2H3细胞,P815细...     

Biological activities of crude plant extracts from Vitex trifolia L. (Verbenaceae)

Biological assays of Vitex trifolia L. organic extracts have shown relevant activities. Hexanic and dichloromethanic (DCM) extracts, when prepared from stems and foliage, have proved to be very toxic against several cancer cell lines in culture (SQC-1 UISO, OVCAR-5, HCT-15 COLADCAR, and KB). Also, an important antifeeding activity against the insect pest Spodoptera frugiperda (Lepidoptera: Noctuidae) was recorded. The hexanic extract from leaves completely inhibited the growth of the fungal plant pathogen Fusarium sp. within the first 2 days of the experiment, but dropped significantly at day 6 (15% inhibition). The potential of V. trifolia for several uses is discussed.     

清开灵、血塞通注射液类过敏反应研究

目的用RBL-2H3细胞初步评价清开灵、血塞通注射液类过敏反应,为完善中药注射剂类过敏反应的筛选提供实验依据。方法 MTT法测定2种药物对细胞生长的IC50;采用不同浓度药物、C48/80或培养液分别刺激细胞30 min,检测上清液中组胺、β-氨基己糖苷酶释放率;另取刺激后的细胞,观察其脱颗粒率及超微结构变化。取ICR小鼠进行类过敏试验,以含伊文思蓝的药物或生理盐水单次尾静脉注射30 min,记录每组耳廓蓝染的动物数、蓝染总耳数及耳廓伊文思蓝渗出量。结果离体实验显示,清开灵、血塞通注射液的IC50均为12.5μL/mL;2种药物在较高浓度均能促进细胞释放组胺和β-氨基己糖苷酶(P0.05,P0.01),呈一定剂量依赖性;形态上可见细胞表面绒毛减少,内部空泡状结构增加。小鼠类过敏试验显示,2种药物对动物耳蓝染率均为100%、伊文思蓝渗出量明显增多(P0.01)。离体与整体实验结果一致。结论清开灵、血塞通注射液具有潜在的致类过敏反应性,RBL-2H3细胞模型用于中药注射剂类过敏性评价具有一定的参考应用价值。     

双黄连注射液对致敏RBL-2H3细胞组胺释放的影响

目的：建立致敏RBL-2H3细胞模型,观察双黄连注射液对其组胺释放的影响,探讨该模型用于体外评价中药注射剂致敏性的可行性。方法：双黄连注射液联合弗氏佐剂免疫Wistar大鼠,收集致敏后大鼠血清,将其与体外培养RBL-2H3细胞孵育12h,建立致敏RBL-2H3细胞模型,双黄连注射液与致敏RBL-2H3细胞共培养1.5h,ELISA法检测培养上清中组胺含量。结果：双黄连注射液致敏大鼠血清IgE水平明显升高,与对照组相比P〈0.05,差异有统计学意义。组胺实验结果显示,双黄连注射液可显著引起致敏RBL-2H3细胞组胺释放,与对照组相比P〈0.05,差异有统计学意义。结论：双黄连注射液可促进致敏RBL-2H3细胞释放组胺,提示RBL-2H3细胞模型具有评价中药注射液所致过敏反应的潜在应用价值     

Inhibitory effects of Acorus calamus extracts on mast cell-dependent anaphylactic reactions using mast cell and mouse model

Ethnopharmacological relevance

Acorus calamus Linn. (Araceae) is a traditional herbal plant used for centuries to treat various allergic symptoms including asthma and bronchitis.

Aim of the study

The present study was focused to provide a pharmacological basis for the traditional use of Acorus calamus in allergic symptoms using the mast cell-dependent anaphylactic reactions in in vitro and in vivo models.

Materials and methods

Cell viabilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Dinitrophenyl-human serum albumin (DNP-HSA) induced β-hexosaminidase and interleukin (IL)-4 productions in IgE-sensitized rat basophilic leukaemia (RBL-2H3) cells were measured by enzymatic assay and enzyme-linked immunosorbent assay (ELISA). Passive cutaneous anaphylaxis (PCA) reaction mouse model was implemented for in vivo studies.

Results

Hot water (HW), butylene glycol (BG), hexane (HE) and steam distilled (SD) extracts of Acorus calamus showed different cytoxicity levels evaluated in RBL-2H3 cells. Sub-toxic doses of HW extract suppressed the β-hexosaminidase secretion and IL-4 production significantly and dose dependently in DNP-HSA induced IgE-sensitized RBL-2H3 cells compared to other extracts of Acorus calamus. Further, in vivo studies also revealed that the HW extract significantly inhibited the PCA reaction in mouse compared to the normal control group.

Conclusion

HW extract of Acorus calamus most effectively inhibited degranulation and IL-4 secretion in DNP-HSA-stimulated RBL-2H3 cells and also reduced the mast cell-mediated PCA reaction in mouse, providing a therapeutic evidence for its traditional use in ameliorating allergic reactions.     

Baicalin and rutin are major constituents in Shuanghuanglian injection involving anaphylactoid reaction

OBJECTIVE: To identify the constituents in Shuanghuanglian injection(SHLI) that correlate with anaphylactoid reaction.METHODS: Chemical fingerprints of 10 batches SHLI samples were determined by High Performance Liquid Chromatography(HPLC), and further investigated by similarity analysis. Combined with optical microscopy, both anaphylactoid experiments and confirmatory assay were displayed in Rat basophil leukemia cells(RBL-2H3) to obtain the histamine release inducing by SHLI. The content of histamine was tested by Enzyme-Linked Immuno Sorbent As-say method. Partial least squares regression(PLSR)method and HPLC-DAD-ESI-MSntechnology were conducted to analyze constituents in SHLI involving anaphylactoid reaction.RESULTS: The results of spectrum and effect relationships showed that the eight constituents were positively correlated with anaphylactoid reaction.Among which, nearly 90% of them were identified as baicalin and rutin with PLSR and HPLC-DAD-ESI-MSn. This result was in accordance with confirmatory assay on RBL-2H3 cells.CONCLUSION: Baicalin and rutin from SHLI were the main constituents involving anaphylactoid reaction.     

Inhibitory effect of various Tunisian olive oils on chemical mediator release and cytokine production by basophilic cells

Tunisian olive oils have been traditionally used as a medicinal food for chronic inflammation. To investigate the antiallergic effect of virgin olive oil samples from five principal olive varieties grown in various regions of Tunisia, we used the type I allergy reaction model using rat basophilic leukemia (RBL-2H3) cells and different dilutions of olive oil samples to determine beta-hexosaminidase release inhibition at two different response stages. Results showed that the Sayali olive oil significantly inhibited beta-hexosaminidase release by the IgE antibody-sensitized, BSA antigen-stimulated RBL-2H3 cells at the antibody-antigen binding stage. The result of our experiment shows that the anti-allergic effect of olive oil at this binding stage may be dependent on their flavone content. The Zarrazi olive oil significantly inhibited beta-hexosaminidase release at the antigen-receptor binding stage. Moreover, we investigated the effect of olive oil samples on histamine release and production of cytokines by activated human basophilic (KU812) cells. Different dilutions of Sayali olive oil dose-dependently inhibited the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4), and different dilutions of Zarrazi olive oil dose-dependently inhibited histamine release and IL-4 production by calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA)-stimulated KU812 cells.     

药用植物蔓荆扦插技术研究

目的探讨蔓荆生根成苗的扦插技术,为大面积种植提供依据。方法利用遮荫小拱棚,以干净的河沙作为扦插基质,选用二年生健壮枝、中度木质化、直径长在1~1.5 cm进行插穗。结果结合药剂处理可获得生根率高、质量优的蔓荆苗木。结论产出的幼苗枝长叶茂,叶色深浓,并且整齐一致。本研究所采用育苗方法简单易行、周期短,可以规模化大批量生产。     

Comparative anti-hyperglycemic potentials of medicinal plants

Validation of the ethnobotanical use of the leaves of Artemisia vulgaris Linn. (Compositae), Eucalyptus tereticornis Sm. (Myrtaceae), Solanum nigrum Linn. (Solanaceae), and Vitex negundo Linn. (Verbenaceae); stems of Nopalea cochinellifera (Linn.) Salm-Dyck (Cactaceae); roots of Imperata cylindrica Beauv. (Gramineae); dried bark of Syzygium cumini (Linn.) Skeels (Myrtaceae) as anti-diabetic agents using the oral glucose tolerance test showed that only the bark of Syzygium cumini and the leaves of Vitex negundo and Eucalyptus tereticornis exhibited anti-hyperglycemic activities when fed simultaneously with glucose. At the same dosages of 5 mg/20 g mouse, Syzygium cumini-treated mice showed a significant decrease in blood glucose levels (BGLs) at 30 min (alpha=0.10) and from 45 min onwards at alpha=0.05. Vitex negundo exhibited greater anti-hyperglycemic activity than Eucalyptus tereticornis. Both showed a significant decrease in BGLs at 60 min but at alpha=0.05 for Vitex negundo and at alpha=0.07 for Eucalyptus tereticornis. There was no significant lowering in BGLs for Imperata cylindrica and Solanum nigrum while there was even an increase in BGLs for Nopalea cochinellifera and Artemisia vulgaris.     

Effects of magnolialide isolated from the leaves of Laurus nobilis L. (Lauraceae) on immunoglobulin E-mediated type I hypersensitivity in vitro

Ethnopharmacological relevance

Laurus nobilis L. (Lauraceae) has been used for folk medicines in the Mediterranean area and Europe to treat various disorders including skin inflammation (dermatitis) and asthma.

Aim of the study

Our aim was to investigate the scientific evaluation of the compounds from Laurus nobilis L. on immuniglobulin E (IgE)-mediated type I hypersensitivity responses in vitro such as atopic dermatitis and asthma.

Methods and materials

Seven compounds were isolated and examined for the mast cell stabilizing effect on IgE-sensitized RBL-2H3 mast cells by measuring the β-hexosaminidase activity. In addition, the effects on interleukin (IL)-4 production and IL-5-dependent Y16 early B cell proliferation were investigated as well as their cytotoxic effects on RBL-2H3 cells.

Results

Among the seven isolated compounds, magnolialide attenuated the release of β-hexosaminidase from RBL-2H3 cells with an IC₅₀ value of 20.2 μM, while the other compounds revealed no significant effects at concentrations tested. Furthermore, magnolialide significantly inhibited the IL-4 release with an IC₅₀ value of 18.1 μM and IL-4 mRNA expression with an IC₅₀ value of 15.7 μM in IgE-sensitized RBL-2H3 cells. In addition, the inhibition of IL-5-dependent proliferation of early B cells (Y16 cells) by magnolialide was demonstrated with an IC₅₀ value of 18.4 μM.

Conclusion

These results suggest that the magnolialide might be a candidate for the treatment of IgE-mediated hypersensitivity responses such as atopic dermatitis and asthma by inhibiting mast cell degranulation, the IL-4 production, and IL-5-dependent early B cell proliferation, key factors in the development and amplification of type I hypersensitivity reactions.     

Anti-allergic activity of some selected plants in the Zingiberaceae family

Ethanolic and water extracts, together with volatile oils from the rhizomes of six selected Zingiberaceous plants, including Curcuma mangga, Kaempferia galanga, Kaempferia parviflora, Zingiber cassumunar, Zingiber officinale and Zingiber zerumbet were investigated for their anti-allergic activities using a RBL-2H3 cell line. The ethanolic (EtOH) extract of Kaempferia parviflora exhibited the most potent anti-allergic effect against antigen-induced beta-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC(50) value of 10.9 microg/ml, followed by Zingiber cassumunar (EtOH, IC(50)=12.9 microg/ml) and Curcuma mangga (water, IC(50)=36.1 microg/ml). The volatile oils of these six plants were apparently inactive (IC(50)>100 microg/ml). The crude extracts were also tested on beta-hexosaminidase activity to clarify whether their effects were due to the inhibition of enzyme activity or of degranulation. As a result, the plant extracts were inactive against the enzyme activity of beta-hexosaminidase. These findings support the use in Thai traditional medicine of these selected Zingiberaceous plants, especially Kaempferia parviflora and Zingiber cassumunar, for treatment of allergy and allergic-related diseases.     

Anti-allergic and anti-inflammatory effects of bakkenolide B isolated from Petasites japonicus leaves

Aims of the study

To elucidate the anti-allergic and anti-inflammatory effects of Petasites genus, we studied the effects of several compounds isolated from Petasites japonicas leaves.

Materials and methods

Bakkenolide B was isolated from Petasites japonicus leaves. Antigen-induced degranulation was measured in RBL-2H3 mast cells by measuring β-hexosamidase activity. Induction of inducible nitric oxide synthase and cyclooxygenase 2 was measured by Western blotting in peritoneal macrophages. Ovalbumin-induced asthma model was used for in vivo efficacy test of bakkanolide B.

Results

We found that bakkenolide B, a major component of the leaves, concentration-dependently inhibited RBL-2H3 mast cell degranulation. Bakkenolide B also inhibited the gene inductions of inducible nitric oxide synthase and cyclooxygenase 2 in mouse peritoneal macrophages. Furthermore, in an ovalbumin-induced asthma model, bakkenolide B strongly inhibited the accumulation of eosinophils, macrophages, and lymphocytes to bronchoalveolar lavage fluid.

Conclusion

Bakkenolide B has suppressive properties for allergic and inflammatory responses and may be utilized as a potent agent for the treatment of asthma.     

Inhibitory effects of adlay bran (Coix lachryma-jobi L. var. ma-yuen Stapf) on chemical mediator release and cytokine production in rat basophilic leukemia cells

Ethnopharmacological relevance

Adlay (Job's tears, Coix lachryma-jobi L. var. ma-yuen Stapf) has long been used in China to treat rheumatism.

Aim of the study

We investigated the anti-allergic effects of adlay bran on rat basophilic leukemia (RBL)-2H3 cells.

Materials and methods

To evaluate the anti-allergic effects of adlay bran, the release of histamines and cytokines were measured using ELISA. To explore the mechanism of these effects, the protein expression levels were determined using western blotting.

Results

A 40.8 μg/mL concentration of the ethyl acetate fraction of the ethanolic extracts of adlay bran (ABE-EtOAc) effectively inhibited mast cell degranulation. The 40–100% EtOAc/Hex subfractions of ABE-EtOAc inhibited histamine release with an IC₅₀ of 71–87 μg/mL. Moreover, the ABE-EtOAc subfractions suppressed the secretion of interleukin (IL)-4, IL-6 and tumor necrosis factor-α in the RBL-2H3 cells, indicating that adlay bran can inhibit cytokine secretion in the late phase of the allergic reaction. In addition, adlay bran reduced the intracellular production of reactive oxygen species, inhibited the phosphorylation of Akt and decreased the expression of protein kinase C. Furthermore, six phenolic acids and one flavone were isolated. Of these compounds, luteolin showed the most potent inhibitory activity (IC₅₀ = 1.5 μg/mL).

Conclusion

Adlay bran extract reduced the release of histamines and cytokines and suppressed the production of Akt. These combined effects influenced the signal transduction in RBL-2H3 cells, thereby revealing the mechanisms of the anti-allergic effects of adlay.     

Inhibitory activity of Chrysanthemi sibirici herba extract on RBL-2H3 mast cells and compound 48/80-induced anaphylaxis

The effects of extracts from various oriental medicinal herbs on mast cell-mediated allergic reaction were investigated. Among them, Chrysanthemi sibirici herba ethanol extract exerted the potent inhibitory activity on antigen-induced degranulation in RBL-2H3 mast cells. Chrysanthemi sibirici herba dose-dependently inhibited DNP-BSA or compound 48/80-induced degranulation in RBL-2H3 mast cells, with IC(50) values of approximately 49 microg/ml and 76 microg/ml, respectively. This extract strongly suppressed compound 48/80-induced systemic anaphylaxis by 48.7% at a dose of 300 mg/kg in mice. Chrysanthemi sibirici herba also inhibited the expression of TNF-alpha and the activation of the MAP kinase, ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of activating phosphorylation of ERK1/2. These results lead us to conclude that Chrysanthemi sibirici herba may be used clinically to treat various allergic diseases.     

Evaluation of the anti-asthmatic property of Asystasia gangetica leaf extracts

The leaf of Asystasia gangetica T. Adams (Acanthaceae) is used in many parts of Nigeria for the management of asthma. This study was aimed at investigating the anti-asthmatic property of hexane, ethylacetate, and methanol extracts of the leaves of Asystasia gangetica, obtained by successive sohxlet extraction. The results indicated that the extracts did not exhibit contractile or relaxant activity in isolated tissue preparations; however, they inhibited the contraction evoked by spasmogens; the IC(50) were calculated, where possible. The extracts relaxed histamine-precontracted tracheal strips in the following degree of potency-ethylacetate extract>hexane extract=methanol extract. The extracts also exhibited anti-inflammatory activity in the order of magnitude-methanol extract>hexane extract>ethylacetate extract. Acute toxicity test estimated an i.p. LD(50) of 2150 mg/kg in mice for methanol extract while phytochemical screening showed the presence of carbohydrates, proteins, alkaloids, tannins, steroidal aglycones, saponins, flavonoids, reducing sugars, and triterpenoids, with the methanol extract having the highest number of constituents. The study justified the use of the leaf of Asystasia gangetica in the management of asthma in Nigerian folk medicine.     

Hystological and pharmacological study of Thymus piperella (L.)

Histological and pharmacological assays have been carried out with methanol, hexane, dichloromethane and butanol extracts of Thymus piperella (L.) leaves. All the extracts were considered innocuo in the toxicity test. Methanol and also hexane, dichlorometane and butanol fractions, inhibited significantly the contractions induced by acetylcholine in isolated rat ileum in a concentration-dependent manner and the hexane extract was the most potent. However, the methanol extract did not modify the contractile effect of noradrenaline and histamine on isolated rat aorta and guinea-pig trachea respectively at the assayed dose (10, 100, 200 microg/mL). These results contribute to explain in part the use of this plant in folk medicine. In addition, morphological and histological structures characteristic of this species have been described for the first time.     

蔓荆子及其不同程度炒制品总黄酮含量测定

目的:测定蔓荆子及其不同程度炒制品中总黄酮的含量。方法: 以芦丁为对照品,采用紫外分光光度法,在500nm处测定吸收度,从而测定蔓荆子各样品中的总黄酮含量。结果:蔓荆子生品、微炒品、炒焦品、炒炭品、炒过炭的总黄酮含量分别为5.68‰、5.85‰、7.89‰、8.02‰、0.31‰。结论:随着蔓荆子炒制程度的加重,总黄酮相对含量呈现先上升后下降的变化,炒制太过总黄酮基本损失殆尽。     

Anti-Helicobacter pylori activity of Aristolochia paucinervis Pomel extracts

The anti-Helicobacter pylori effect of the extracts and the fractions obtained from Aristolochia paucinervis rhizome and leaves were studied against a reference strain of H. pylori by using the agar dilution method. Only the methanol extracts and the hexane fractions of either the rhizome or the leaves exhibited an inhibitory activity at a concentration of < or =128 microg/ml. The leaf hexane fraction APLH demonstrated a higher inhibitory activity (MIC: 4 microg/ml) than the rhizome hexane fraction APRH (MIC: 16 microg/ml), the leaf methanol extract APLM (MIC: 32 microg/ml) and the rhizome methanol extract APRM (MIC: 128 microg/ml). This inhibitory activity was confirmed for the active extracts and fractions against clinical isolates of H. pylori (n = 20) for which MIC50) and MIC90 were determined.