中药注射剂中黄芩苷致类过敏反应与hMRGPRX2受体关系研究

Baicalin, the main component of Scutellaria baicalensis, is widely existed in traditional Chinese medicine injection (TCMI), yet the adverse reactions to this injection have been reported by mutiple studys, most of them are anaphylactic reactions. Baicalin was screened from Shuanghuanglian injection by using RBL-2H3/CMC model in our previous study. In vivo and in vitro studys showed that baicalin induced degranulation of mast cell and increased mouse capillary permeability, which has the potential allergenic effect. Since hMRGPRX2 has been found as the key targets of anaphylactoid reaction in recent studies, it is of vital importance to inverstigate the relationship between baicalin induced anaphylactoid reaction and hMRGPRX2. Based on the selection of hMRGPRX2 as the target and the methods of molecular biology and pharmacology, our study intend to investigate the activation of baicalin on mastocyte, and analyze the dose-response relationship between anaphylactoid reaction and baicalin. We will build high and low expression hMRGPRX2 cells，establish CMC model，combine with Frontal and thermodynamic analysis, radioligand binding assay, cell degranulation assay and hMRGPRX2 signal transduction analysis, to illuminates the anaphylactoid inducement mechanism of baicalin via hMRGPRX2 preliminarily. The completion of projects provide experimental basis for clinical safety use of baicalin, anaphylactoid components screening model from TCMI establishment will provide new ideas in TCMI anaphylactoid study.

黄芩苷是临床常用中药注射剂配伍药材黄芩的主成分，此类注射剂不良反应报道较多，大部分为类过敏反应。课题组前期用RBL-2H3细胞膜色谱模型（CMC）从双黄连注射液中筛选出黄芩苷，发现黄芩苷造成肥大细胞脱颗粒、小鼠毛细血管通透性增加，具有潜在致敏性。hMRGPRX2是近期发现的类过敏关键靶点，研究黄芩苷类过敏效应与该受体关系有重要意义。本课题以hMRGPRX2为靶点，利用分子生物学、药理学等方法研究黄芩苷对肥大细胞激活效应，分析黄芩苷致类过敏反应与剂量关系；建立hMRGPRX2过表达及沉默细胞模型，通过hMRGPRX2/CMC模型、前沿及热力学分析、放射性配体受体结合分析、脱颗粒实验、hMRGPRX2信号转导分析，初步阐明黄芩苷与hMRGPRX2关系及致类过敏分子机理。项目的完成为黄芩苷临床安全使用提供实验依据，同时建立中药注射剂致类过敏成分筛选模型，为中药注射剂致类过敏反应研究提供新的思路。

黄芩苷是临床常用中药注射剂配伍药材黄芩的主成分，此类注射剂不良反应报道较多，大部分为类过敏反应。课题组用RBL-2H3细胞膜色谱模型（CMC）从双黄连注射液中筛选出黄芩苷，发现黄芩苷造成肥大细胞脱颗粒、小鼠毛细血管通透性增加，具有潜在致敏性。本项目选用balb/c小鼠耳廓血管渗透性实验（小鼠蓝耳实验）及C57小鼠脚趾肿胀实验为致敏评价模型。当黄芩苷静脉注射给药剂量为262.1 mg/kg时，小鼠出现过敏症状，此时换算为人的静脉注射剂量为28.9 mg/kg。因此，拟定人当日单次静脉注射黄芩苷的量不宜超过28.9 mg/kg；脱颗粒实验结果表明，当黄芩苷浓度达到50 μM时，可引起RBL-2H3细胞发生脱颗粒反应，当黄芩苷浓度为100 μM时，其引发脱颗粒反应能力同阳性对照C48/80相当。因此，该实验结果说明黄芩苷导致脱颗粒反应发生的最低浓度为50 μM；利用基因干扰技术沉默RBL-2H3细胞中MRGPRB3（大鼠源，与人源MRGPRX2同源）后，黄芩苷对该细胞无激活作用；此外，黄芩苷可以增加RBL-2H3细胞胞质内游离Ca2+浓度，且呈剂量依赖性。黄芩苷还可剂量刺激MRGPRX2高表达HEK293细胞钙离子释放，引起胞内钙离子浓度升高。而在正常HEK293细胞内黄芩苷刺激并无此作用。基于以上的体外药理学实验结果得出黄芩苷当日单次静脉注射摄入量不宜超过28.9 mg/kg。建立的MRGPRX2筛选系统，筛选出多种过敏和抗过敏成分。为后续的药物研发提供基础。