紫花地丁止痒复方对RBL-2H3细胞脱颗粒的影响

目的考察紫花地丁止痒复方(Viola yedoensis makino antiitching compound,VYAC)对RBL-2H3细胞脱颗粒的影响及机制。方法CCK-8检测VYAC对RBL-2H3细胞的毒性;C48/80诱导RBL-2H3细胞发生脱颗粒,台盼蓝染色、β-氨基己糖胺酶释放、组胺释放、细胞内Ca2+浓度,评价VYAC对C48/80诱导的RBL-2H3细胞脱颗粒情况;Western blot法检测相关蛋白(Syk、p-Syk、PI3K、Akt、p-Akt)的表达。结果100 mg·L^-1的C48/80能够明显刺激RBL-2H3细胞发生脱颗粒,脱颗粒率达74%(P<0.05);VYAC呈剂量依赖性抑制β-氨基己糖胺酶和组胺的释放(P<0.05),且剂量在800 mg·L^-1以下时不影响RBL-2H3细胞存活;VYAC能够明显减弱细胞内荧光强度,降低细胞内Ca2+浓度;VYAC(25、100 mg·L^-1)明显抑制PI3K蛋白表达、抑制Syk、Akt蛋白的磷酸化(P<0.05)。结论VYAC能够抑制过敏性皮炎中肥大细胞脱颗粒,抑制Ca2+内流,其机制可能是抑制Syk/PI3K/Akt活化。     

基于实时细胞分析技术评价注射用益气复脉(冻干)类过敏反应

目的 建立中药注射剂体外类过敏反应评价方法,快速评价不同批次注射用益气复脉（冻干）类过敏反应表现。方法 体外培养嗜碱性白血病细胞株RBL-2H3细胞,选择Compound 48/80为阳性药,采用实时细胞分析（real-time cell analysis,RTCA）系统检测药物干预后引起的细胞指数（CI）值变化,并利用甲苯胺蓝、鬼笔环肽染色观察细胞形态、骨架变化,以及检测组胺和β-已糖苷酶释放量验证RBL-2H3细胞的脱颗粒情况。选择20个批次的注射用益气复脉（冻干,100 μg/mL）作用于RBL-2H3细胞,进行基于RTCA技术的类过敏反应评价。结果 阳性药Compound 48/80（20 μg/mL）能够使RBL-2H3细胞的CI值在加药后30 min内呈先快速上升后下降趋势;形态学研究发现,Compound 48/80使细胞形态和细胞骨架均发生明显改变,发生明显的脱颗粒现象;组胺和β-己糖苷酶释放实验进一步证实Compound 48/80导致炎症介质的释放,引起了明显的脱颗粒现象;提示RTCA系统可以用于快速敏感的评价RBL-2H3细胞脱颗粒。不同批次的注射用益气复脉（冻干）对RBL-2H3细胞CI值无明显影响,提示所选批次为合格批次,无类过敏反应现象的发生。结论 建立了一套基于RTCA系统的类过敏反应体外快速评价技术,可用于注射用益气复脉（冻干）等中药注射剂类过敏反应的体外快速评价。     

非免疫性过敏反应细胞模型的建立

目的:通过非免疫性刺激物C48/80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2H3细胞脱颗粒反应正交试验,优化非免疫性过敏反应细胞模型建立条件.方法:在不同的孵育时间下,C48/80与不同浓度的RBL-2H3细胞共孵育,通过测定β-氨基己糖苷酶的释放率确定建立非免疫型过敏反应细胞模型的最优实验条件,并且分析不同检测方法间的差异.结果:不同浓度、不同孵育时间下的RBL-2H3细胞均可受C48/80诱导发生典型的脱颗粒反应,但不同条件下的脱颗粒程度和β-氨基己糖苷酶释放率都有显著差异.结论:当RBL-2H3细胞浓度为2×105 mL、孵育时间60 min时,细胞的感受性较好,其脱颗粒程度与药物浓度呈正相关.     

参麦注射液致RBL-2H3细胞脱颗粒的研究

目的 探讨参麦注射液(SMI)对RBL-2H3细胞脱颗粒的影响及其原因。方法 以C48/80为工具药建立RBL-2H3细胞脱颗粒模型, 检测不同浓度C48/80与RBL-2H3细胞作用不同时间后细胞β-己糖苷酶、类胰蛋白酶和组胺的释放率以及细胞活力, 在细胞活力大于80%情况下, 选择释放程度较高的指标和条件为优选考察指标和条件。将SMI和其溶剂(Tween-80)原液等比稀释成不同浓度后与RBL-2H3细胞共同培养, 通过中性红染色法观察细胞脱颗粒的形态学变化, 分别用显色法和间接荧光法检测细胞上清的β-己糖苷酶和组胺释放率, 采用细胞计数法(CCK-8)检测细胞的活力。结果 RBL-2H3细胞脱颗粒模型的最佳作用时间为30 min, 最佳指标为β-己糖苷酶和组胺释放率。与空白组相比, SMI质量浓度低于13.3 g生药/L(3倍临床浓度)时, 细胞中性红染色未见脱颗粒现象, 组胺和β-己糖苷酶释放率亦无差异;而在Tween-80质量浓度为1.00 g/L时, SMI 40 g生药/L(9倍临床浓度)组和溶剂1.00 g/L组细胞中性红染色均可见脱颗粒现象, 细胞上清组胺和β-己糖苷酶释放率亦明显增加。此外, CCK-8结果显示, 与空白组相比, 各浓度的SMI对细胞活力均无影响。结论 SMI低于3倍临床浓度无明显RBL-2H3细胞脱颗粒作用;而在9倍临床浓度能刺激细胞脱颗粒, 这种脱颗粒作用可能与所含溶剂(Tween-80)有关, 与其对RBL-2H3的细胞毒性作用无关。提示SMI在低于3倍临床浓度相对安全, 在9倍临床浓度时有致类过敏反应的风险。     

清开灵注射液类过敏反应应急检验方法探讨

目的：探讨清开灵注射液类过敏反应应急检验方法。方法：将40只ICR小鼠随机均分为阳性对照组、空白组及清开灵高、中、低剂量组,一次性尾静脉注射清开灵注射液,观察清开灵注射液对RBL-2H3细胞β-氨基己糖苷酶及组胺释放率的影响。结果：清开灵注射液可明显增加RBL-2H3细胞β-氨基己糖苷酶及组胺释放率,且呈浓度依赖性。结论：ICR小鼠体内类过敏反应检查法联合RBL-2H3细胞β-氨基己糖苷酶及组胺释放率检查法可作为清开灵注射液类过敏反应应急检验方法。     

中药注射剂所含吐温-80与过敏反应关系的研究

目的：观察不同浓度吐温-80溶液、吐温-80含量不同的中药注射剂对RBL-2H3细胞脱颗粒的影响,探讨中药注射剂所含吐温-80与过敏反应的关系。方法：体外培养RBL-2H3细胞,加入不同浓度（40、20、10、2、1、0.2、0.1、0.05mg/mL）的吐温-80溶液,之后加中性红染液,计数不同浓度吐温-80溶液各组及对照组的脱颗粒细胞,并计算其百分率,同时检测细胞上清液中β-氨基己糖苷酶及组胺的释放量;测定穿琥宁注射液和香丹注射液中吐温-80的含量,2种中药注射剂对RBL-2H3细胞的半数抑制浓度（IC50）以及加入2种注射液各组细胞释放组胺的量。结果：中性红染色实验显示吐温-80可导致RBL-2H3细胞脱颗粒,表现为肥大细胞体积变大,内有空泡产生;浓度为40、20、10、2、1、0.2、0.1mg/mL的吐温-80溶液各组和RPMI1640对照组导致细胞的脱颗粒百分率分别为（57.38±0.47）、（32.54±2.33）、（21.74±0.72）、（16.96±0.26）、（11.40±1.70）、（9.71±0.26）、（7.22±0.15）和（1.51±1.39）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;浓度为40、20、2、1、0.2mg/mL的吐温-80溶液各组和RPMI1640对照组致细胞β-氨基己糖苷酶的释放率分别为（52.44±1.53）、（18.91±0.77）、（7.50±1.82）、（6.65±0.20）、（6.15±0.27）和（0.35±0.06）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;不同浓度吐温-80溶液引起RBL-2H3细胞释放组胺的量也不同;当吐温-80溶液浓度为20～0.1mg/mL时,RBL-2H3细胞脱颗粒百分率、β-氨基己糖苷酶的释放率及其释放组胺的量均与吐温-80溶液的浓度呈线性关系（r=0.9862,r=0.9849,r=0.9740）。穿琥宁注射液和香丹注射液中吐温-80的含量分别为（0.086±0.004）和（0.070±0.008）mg/mL,2种注射液对RBL-2H3细胞的IC50分别为（57.4±1.2）、（1.0±0.2）μL/mL,穿琥宁注射液和香丹注射液组组胺释放量分别为（2.39±0.01）和（1.87±0.00）ng/mL。结论：吐温-80可引起RBL-2H3细胞脱颗粒释放炎症介质;RBL-2H3细胞组胺的释放量与中药注射剂中吐温-80的含量有关;中药注射剂中所含的吐温-80可能与过敏反应的发生有关。     

聚山梨酯-80致RBL-2H3肥大细胞脱颗粒的研究

目的:研究聚山梨酯-80对RBL-2H3肥大细胞脱颗粒释放组胺的影响.方法:培养大鼠来源的RBL-2H3肥大细胞,取不同厂家来源的聚山梨酯-80与RBL-2H3细胞共培养60 min,用荧光分光光度法定量检测RBL-2H3细胞释放的组胺量,计算组胺释放率.结果:不同厂家来源的聚山梨酯-80与RBL-2H3细胞作用60 min后,细胞的组胺释放率与空白对照组相比均显著增加,在一定浓度范围内,组胺的释放随聚山梨酯-80浓度的增加而增加.结论:聚山梨酯-80可导致RBL-2H3肥大细胞脱颗粒,并存在着明显的量效关系,为研究聚山梨酯80致过敏反应机制提供了一定的依据.     

药用注射辅料聚山梨酯80诱发类过敏反应的细胞研究

目的:观察聚山梨酯80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2 H3细胞脱颗粒现象,探讨其诱发类过敏反应的可能机制。方法:聚山梨酯80与RBL-2 H3细胞共同孵育60 min后,透射电镜观察细胞发生脱颗粒反应的形态学变化,荧光显微镜下观察细胞膜磷脂酰丝氨酸与Annexin V结合变化,分光光度法测定细胞上清液β-氨基己糖苷酶释放百分率,并通过阿利新蓝染色试验进行定性观察。结果:不同浓度聚山梨酯80可诱导细胞发生典型的脱颗粒反应形态学变化,与阴性对照组相比脱颗粒率及组胺释放率显著升高,且具有浓度依赖性。结论:聚山梨酯80单次、首次给药即可诱导RBL-2 H3细胞脱颗粒,释放组胺,诱发类过敏反应,这可能是其临床首次用药即引起严重不良反应的机制之一。     

聚山梨酯80刺激肥大细胞RBL-2H3脱颗粒作用的评价

目的：研究不同类别及厂家的聚山梨酯80直接刺激肥大细胞RBL-2H3脱颗粒的作用,为建立完善的注射用辅料引发类过敏反应的筛选评价体系提供依据。方法：以不同剂量的聚山梨酯80处理RBL-2H3细胞,测定β-氨基己糖苷酶释放量,并通过MTT法进一步研究有脱颗粒作用的受试物对RBL-2H3细胞的毒性作用。结果：8种聚山梨酯80样品具有不同程度的直接刺激RBL-2H3细胞脱颗粒的作用,且呈浓度依赖性,其中上海试剂采购供应站提供的受试物作用极强,威尔制药口服级受试物作用较强,威尔制药A、威尔制药B、威尔制药注射级、威尔制药4个受试物作用相近,2个进口品种的作用略弱。在产生直接刺激RBL-2H3细胞脱颗粒作用的浓度下,上海试剂采购供应站的聚山梨酯80有明显的细胞毒性,并呈浓度依赖性。结论：聚山梨酯80能够直接刺激RBL-2H3细胞脱颗粒,其作用强度不同反映了产品的质量差异;上海试剂采购供应站提供的聚山梨酯80直接刺激RBL-2H3细胞脱颗粒的作用可能由其细胞毒性引起。     

虾原肌球蛋白及其IgE单克隆抗体建立I型过敏反应体内外模型

目的利用虾原肌球蛋白及其Ig E单克隆抗体建立I型过敏反应体内外模型。方法采用等电点沉淀法制备刀额新对虾原肌球蛋白,利用杂交瘤技术制备抗虾原肌球蛋白Ig E单克隆抗体。建立虾原肌球蛋白攻击Ig E单抗致敏的RBL-2H3细胞模型,测定β-氨基己糖苷酶释放量。利用虾原肌球蛋白及其Ig E单抗建立被动全身过敏反应小鼠模型,监测抗原攻击后30 min内肛温的变化。结果 Ig E单抗致敏的RBL-2H3细胞在虾原肌球蛋白攻击后发生脱颗粒,β-氨基己糖苷酶释放量明显增加。虾原肌球蛋白及其Ig E单抗诱导了小鼠被动全身过敏反应,抗原攻击后小鼠肛温平均下降约1.44℃。结论虾原肌球蛋白及其Ig E单克隆抗体成功建立了I型过敏反应体内外模型。     

Imiquimod-related dermatitis is mainly mediated by mast cell degranulation via Mas-related G-protein coupled receptor B2

While imiquimod (IMQ) has been widely used in dermatology, its side effect manifested as dermatitis couldn't be ignored. However, the underlying mechanism has not been fully understood. Considering the clinical features of IMQ-related dermatitis similar to pseudo-allergic reaction and the presence of large numbers of mast cell in tissues treated with IMQ, the possibility that IMQ-related dermatitis mediated by mast cell-specific Mas-related G protein-coupled receptor X2 (MRGPRX2) should be addressed. To investigate the role of MRGPRX2 in vivo, MrgprB2, the mice homology of human MRGPRX2, was detected in IMQ-induced dermatitis mouse model. Histopathological changes including mast cell degranulation and footpad swelling were assayed in wild-type and MrgprB2^−/− mice. The results showed that IMQ application induced dermatitis and footpad swelling with inflammatory cells infiltration plus mast cell activation in the skin of wild-type mice but reduced significantly in MrgprB2^−/− mice. Further, compared to wild-type mice, serum histamine and inflammatory cytokine levels were compromised in MrgprB2^−/− mice treated with IMQ, while the serum IgE level didn’t change significantly. In vitro studies, levels of mediators released from murine peritoneal mast cells (MPMCs) after IMQ treatment were increased in a dose-dependent manner, which were much mild in MPMCs from MrgprB2^−/− mice. Intracellular Ca²⁺ concentration was increased in a dose dependent manner after IMQ treatment both in MrgprB2-HEK293 and MRGPRX2-HEK293 cells. Moreover, β-hexosaminidase released after IMQ treatment was blocked by siRNA directed at the MRGPRX2 receptor in LAD2 cells. In summary, MrgprB2 /MRGPRX2 mediate mast cell activation and participate in IMQ-related dermatitis.     

Mast cell degranulation induced by chlorogenic acid

Aim:

To investigate the mechanism of chlorogenic acid (CA)-induced anaphylactoid reactions.

Methods:

Degranulation of peritoneal mast cells was assayed by using alcian blue staining in guinea pigs, and the degranulation index (DI) was calculated. CA-induced degranulation of RBL-2H3 cells was also observed and assayed using light microscopy, transmission electron microscopy, flow cytometry, and β-hexosaminidase release.

Results:

CA 0.2, 1.0, and 5.0 mmol/L was able to promote degranulation of peritoneal mast cells in guinea pigs in vitro, but it did not increase the degranulation of peritoneal mast cells in CA-sensitized guinea pigs compared with control (P>0.05). Treatment with CA 0.2, 1.0, and 5.0 mmol/L for 30, 60, and 120 min induced degranulation in RBL-2H3 cells in a dose- and time-dependent manner (P<0.01). Under transmission electron microscope typical characteristics of degranulation, including migration of granular vesicles toward the plasma membrane and integration combined with exocytosis, were observed, after CA or C48/80 treatment. Fluorescent microscopy and flow cytometric analysis showed that CA induced concentration-dependent translocation of phosphatidylserine in RBL-2H3 cells. β-hexosaminidase release in RBL-2H3 cells was significantly increased after incubation with 1 mmol/L CA for 60 min and 5 mmol/L CA for 30 min (P<0.01).

Conclusion:

CA induces degranulation of peritoneal mast cells and RBL-2H3 cells in guinea pigs, which might be one of the mechanisms of the generation of anaphylactoid reactions induced by CA.     

Quercetin inhibits Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R related Ca2+ fluctuations

An allergic reaction is a potentially fatal hypersensitivity response caused by mast cell activation, particularly histamine and lipid mediators. Histamine release caused by reaction to drugs is considered a pseudo-allergic reaction. Quercetin is known for its anti-allergic immune effect. However, at present, its anti-pseudo-allergic effect and its mechanism are less investigated. Therefore, the purpose of this study was to evaluate the anti-pseudo-allergic effect of Quercetin in vivo and to explore the mechanism in vitro. The anti-pseudo-allergic activity of Quercetin was evaluated in vivo using a mouse model, while Quercetin mechanism of action was examined in vitro using HEK293 cells expressing Mrgprx2, a mast cell specific receptor, and LAD2 mast cell line. Our in vivo results showed that Quercetin could attenuate Evans blue leakage in the paws and hind paw thickness in C57BL/6 mice in a dose-dependent manner, and could significantly inhibit serum histamine and chemokines release. In addition, it suppressed calcium mobilization and attenuated the release of histamine and MCP-1 in peritoneal mast cells in a dose-dependent manner. Furthermore, it inhibited the vasodilation due to histamine, the release of eosinophils, and the percentage of degranulated mast cells, indicating that Quercetin antagonized mast cell mediators in vivo, histamine-induced vasodilation and eosinophil release.In vitro results showed that Quercetin reduced pseudo-allergic induced calcium influx, suppressed degranulation and chemokines release in a similar way as dexamethasone (100 μM) (mast cell stabilizer) in LAD2 mast cell line. In addition, Quercetin inhibited Mrgprx2-induced both calcium influx and pseudo-allergic reaction in HEK293 cells expressing Mrgprx2. C48/80, a histamine promoter, and Substance P (a neuropeptide) EC₅₀ was higher when combined with Quercetin compared to the EC₅₀ of these compounds alone, suggesting that Quercetin could inhibit Mrgprx2-induced pseudo-allergic reaction. Furthermore, Quercetin decreased PLCγ-IP3R signaling pathway activation induced by C48/80 in LAD2 mast cell line. In Mrgprx2 knockdown LAD2 cells, the effect of Quercetin (200 μM) reduced C48/80 induced calcium flux and the release of β‑hexosaminidase, histamine, MCP-1 and IL-8 compared with non-atopic control (NC) transfected LAD2 human mast cells, suggesting that Quercetin anti-pseudo-allergic effect was related to Mrgprx2. The docking results showed that Quercetin had a good binding affinity with Mrgprx2 similar to the one of Substance P and C48/80. Therefore, Quercetin inhibited Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca²⁺ fluctuations.Our results validated Quercetin as an effective small molecule inhibiting Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca²⁺ fluctuations, thus highlighting a potential candidate to suppress Mrgprx2 induced pseudo-allergic related diseases.     

Vancomycin-induced release of histamine from rat peritoneal mast cells and a rat basophil cell line (RBL-1).

Rapid intravenous administration of the glycopeptide antibiotic, vancomycin, may cause a hypotensive reaction which can usually be prevented by infusing vancomycin in dilute solutions. The release of histamine from circulating cells such as basophils and tissue mast cells has been implicated in hypotensive reactions since the effects can be prevented by antihistamine pretreatment. The direct effects of vancomycin on histamine release were therefore investigated in rat peritoneal mast cells and rat leukemic basophils (RBL-1 cells). Suspension cultures of mast cells or RBL-1 cells were exposed to vancomycin for 30-60 minutes at concentrations comparable to those infused clinically (2.28 or 4.56 mg/ml). Vancomycin induced a time- and dose-dependent release of histamine into the culture media from both cell types. The reference degranulating agent, Compound 48/80 (CP 48/80), was also shown to induce histamine release from mast cells and RBL-1 cells. Mast cells were significantly more sensitive to vancomycin and CP 48/80 than RBL-1 cells and, unlike RBL-1 cells, were responsive to the inhibitory effects of cromolyn sodium on histamine release. Cromolyn sodium did not inhibit vancomycin-induced histamine release in RBL-1 or mast cells. Morphologically, mast cells exposed to either vancomycin or CP 48/80 exhibited dose-related degranulation. On the other hand, treatment-related degranulation effects of either vancomycin or CP 48/80 on RBL-1 cells could not be reliably distinguished from controls by qualitative evaluation. Based upon these findings it is concluded that mast cells may represent a more useful model to evaluate the potential of investigational agents to release histamine and to study mechanisms of histamine release than RBL-1 cells.     

G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: roles of Mas-related genes MrgX1 and MrgX2

Although human mast cells express G protein coupled receptors for the anaphylatoxin C3a, previous studies indicated that C3a causes mast cell degranulation, at least in part, via a C3a receptor-independent mechanism similar to that proposed for polycationic molecules such as compound 48/80. The purpose of the present study was to delineate the receptor specificity of C3a-induced degranulation in human mast cells. We found that C3a, a C3a receptor "superagonist" (E7) and compound 48/80 induced Ca(2+) mobilization and degranulation in a differentiated human mast cell line, LAD2. However, C3a and E7 caused Ca(2+) mobilization in an immature mast cell line, HMC-1 but compound 48/80 did not. We have previously shown that LAD2 cells express MrgX1 and MrgX2 but HMC-1 cells do not. To delineate the receptor specificity for C3a and compound 48/80 further, we generated stable transfectants expressing MrgX1 and MrgX2 in a rodent mast cell line, RBL-2H3 cells. We found that compound 48/80 caused degranulation in RBL-2H3 cells expressing MrgX1 and MrgX2 but C3a did not. By contrast, E7 activated RBL-2H3 cells expressing MrgX2 but not MrgX1. These findings demonstrate that in contrast to previous reports, C3a and compound 48/80 do not use a shared mechanism for mast cell degranulation. It shows that while compound 48/80 utilizes MrgX1 and MrgX2 for mast cell degranulation C3a does not. It further reveals the novel finding that the previously characterized synthetic peptide, C3a receptor "superagonist" E7 activates human mast cells via two mechanisms; one involving the C3a receptor and the other MrgX2.     

Isolation of 5-(hydroxymethyl)furfural from Lycium chinense and its inhibitory effect on the chemical mediator release by basophilic cells

The effect of hot water extracts of LYCIUM CHINENSE fruits (LCF) on the β-hexosaminidase (β-hexo) release by IgE sensitized BSA stimulated rat basophilic leukemia (RBL-2H3) cells was investigated. The ethylacetate (EtOAc) layer of the extract has shown an inhibitory effect on β-hexo release from RBL-2H3 cells at the antigen antibody binding stage. The water (H?O) fraction (EFW) of the chloroform (CHCl?) extract from the EtOAc layer also inhibited β-hexo release at the same stage in a dose-dependent manner. With column chromatography preparation, proton and carbon nuclear magnetic resonance (1H and 13C NMR) spectra, electron ionization mass spectrometer (EI-MS) spectra, and high-performance liquid chromatography (HPLC) analysis, the active component was determined to be 5-(hydroxymethyl)furfural (5-HMF). Thus, the 5-HMF showed an inhibitory effect on β-hexo release at the antigen-antibody binding stage and the antibody-receptor binding stage. Furthermore, 5-HMF suppressed [Ca2+] I influx in the IgE-sensitized BSA-stimulated RBL-2H3 cells. Our results show that 5-HMF may be useful for the treatment or prevention of type I allergic diseases.