基于RBL-2H3细胞和ICR小鼠过敏与类过敏叠加模型评价注射用血塞通(冻干)过敏与类过敏反应  

An experimental study of combination of allergic and anaphylactoid reactions to Xuesaitong Injection(freeze-drying)based on RBL-2H3 cells and ICR mice model

作　　者：刘方美 张羽[1] 汤书婉 符胜男 冷雪娇[1] 李存玉[1,2,3] 郑云枫 彭国平[1,2,3] LIU Fangmei;ZHANG Yu;TANG Shuwan;FU Shengnan;LENG Xuejiao;LI Cunyu;ZHENG Yunfeng;PENG Guoping(College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,Nanjing 210023,China;National Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture,Lianyungang 222047,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]江苏省中药资源产业化过程协同创新中心,江苏南京210023 [3]中药制药过程控制与智能制造技术全国重点实验室,江苏连云港222047

出　　处：《药物评价研究》2024年第5期982-993,共12页Drug Evaluation Research

基　　金：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)。

摘　　要：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(30μg·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�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 theβ-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 andβ-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-

关 键 词：过敏反应 类过敏反应 叠加 RBL-2H3 ICR小鼠 注射用血塞通(冻干) 

分 类 号：R965.3[医药卫生—药理学]