目的 建立过敏和类过敏叠加的RBL-2H3细胞和ICR小鼠动物模型,对注射用血塞通(冻干)(XST)的过敏与类过敏反应进行评价研究。方法 体外以RBL-2H3细胞的β-氨基己糖苷酶(β-Hex)和组胺释放率为评价指标,确定抗二硝基苯单克隆抗体(DNP-IgE)、DNP-牛血清白蛋白(BSA)的剂量及与C48/80(30 μg·mL^-1)作用的最佳时间,筛选过敏和类过敏叠加模型的阳性条件,随后考察XST (4、8、16 mg·mL^-1)对细胞活力及与DNP-IgE/BSA叠加后对细胞脱颗粒的影响。体内以ICR小鼠为实验对象,以(类)过敏反应症状分值及血浆中免疫球蛋白E (IgE)、组胺、5-羟色胺、血管内皮生长因子A (VEGF-A)、末端补体复合物(SC5b-9)含量为评价指标,筛选卵蛋白(OVA,2.5、5.0、10.0 mg·kg^-1)、C48/80(1、2、4 mg·kg^-1)过敏-类过敏模型阳性条件,最后对XST (60、120、240 mg·kg^-1)的致敏性及是否会产生过敏、类过敏反应进行评价。结果 体外细胞实验最终确定400 ng·mL^-1的DNP-IgE致敏后用50 ng·mL^-1的DNP-BSA激发的同时与C48/80共同作用30 min作为过敏与类过敏叠加阳性组;16 mg·mL^-1的XST与DNP-IgE/BSA联合叠加时,与单给DNP-IgE/BSA或XST组比较均促进组胺和β-Hex的释放(P<0.01)。体内小鼠实验中5、10 mg·kg^-1的OVA均会使小鼠体内IgE显著升高,依据过敏样反应分值和小鼠血浆内组胺、VEGF-A和SC5b-9含量最终确定5 mg·kg^-1 OVA与1 mg·kg^-1 C48/80建立叠加模型,耳、肺及支气管组织中可见明显的水肿及炎性细胞浸润。单纯的XST不会对小鼠致敏,但是与OVA介导的过敏反应叠加后,与单给DNP-IgE/BSA或XST组比较,会显著提高血浆中内组胺、VEGF-A和SC5b-9水平(P<0.05、0.01),与建立的过敏-类过敏叠加模型表现出较好地一致性。结论 体外体内实验均表明IgE介导的过敏反应和C48/80引起的类过敏反应会产生叠加作用,加剧过敏介质的释放和免疫反应程度。同时此模型的建立也验证了XST存在过敏与类过敏叠加现象,该模型可为中药注射剂的临床前安全性评价及合理用药提供参考。

Objective To establish an allergic and anaphylactoid superimposed reaction RBL-2H3 cells and ICR mice model for the study of allergic and anaphylactoid reaction to Xuesaitong Injection (freeze-drying) (XST). Methods This study employed the β -hexosidase and histamine release rate of RBL-2H3 cells as evaluation indices in vitro to determine the optimal dose of DNPIgE, DNP-BSA, and the duration of interaction with C48/80. The study investigated the effects of various doses of XST on cell viability and degranulation after stacking. In vivo, ICR mice were used as experimental subjects, and the scores of (type) allergic reaction symptoms and the contents of immunoglobulin E (IgE), histamine, 5-hydroxytryptamine, vascular endothelial growth factor A (VEGF-A), and terminal complement complex (SC5b-9) in plasma were used as evaluation indicators to screen positive conditions for OVA (2.5, 5.0, 10.0 mg·kg^-1) and C48/80 (1, 2, 4 mg·kg^-1) allergy-like allergic models, and finally, the sensitization of XST (60, 120, 240 mg·kg^-1) and whether it would cause allergic or allergic-like reactions were evaluated. Results The positive group for anaphylactic-anaphylactoid superimposed reaction was determined to be 400 ng·mL^-1 DNP-IgE sensitised with 50 ng·mL^-1 DNPBSA and co-administered with C48/80 for 30 min. Additionally, the release of His and β-Hex was promoted by 4, 8, and 16 mg·mL^-1 of XST in combination with IgE. In the in vivo mouse experiment, 5 and 10 mg·kg^-1 OVA significantly increased IgE levels in mice. Based on the allergy-like reaction score and the contents of histamine, VEGF-A, and SC5b-9 in the plasma, the 5 mg·kg^-1 OVA and 1 mg·kg^-1 C48/80 were used to establish a stacked model of allergy-like reaction. There was obvious edema and inflammatory cell infiltration in the ear, lung, and bronchial tissue. XST alone did not sensitize mice, but when combined with OVA-induced allergic reaction, it significantly increased the levels of intra-plasma histamine, VEGF-A, and SC5b-9 compared to the single administration of DNP-IgE/BSA or XST groups (P < 0.05, 0.01), showing good consistency with the established allergy-like-allergy stacked model. Conclusion In vitro and in vivo experiments have shown that IgE-mediated allergic reactions and C48/80-induced anaphylactoid reactions produce a superimposed effect, exacerbating the release of allergic mediators and the degree of immune response. The establishment of this model also verified the existence of allergic and anaphylactoid superposition phenomenon of XST. This model can provide a reference for the preclinical safety evaluation and rational use of Chinese medicine injection.

R965.3

国家自然科学基金资助项目(82074006);江苏省自然科学基金面上项目(BK20211303)