注射用双黄连诱发肥大细胞脱颗粒的早期生物学效应

Early biological effects of mast cell degranulation induced by Shuanghuanglian injection

目的以体外培养的肥大细胞为实验材料,考察注射用双黄连诱发肥大细胞脱颗粒早期的生物学效应,初步探讨中药注射剂安全性评价的质控指标。方法肥大细胞为RBL-2H3细胞株(ATCC-6562),注射用双黄连购自哈药集团中药二厂(Z20043425);细胞活力检测采用MTT法,β-氨基己糖苷酶检测采用ELISA法;采用扫描电镜观察肥大细胞形态,用激光共聚焦显微镜检测活细胞内钙离子浓度。结果注射用双黄连细胞毒性剂量及致类过敏剂量为≥0.08 mg/m L;肥大细胞脱颗粒反应出现在药物作用早期(≤1分钟);较低浓度注射用双黄连诱发肥大细胞内钙离子浓度峰值出现在4分钟左右。结论注射用双黄连作用肥大细胞早期,可激活Ca^(2+)相关信号通路、启动肥大细胞脱颗粒反应、大量释放β-氨基己糖苷酶。该结果提示:基于RBL-2H3细胞的Ca^(2+)浓度及致敏介质检测平台有望成为中药注射剂安全性评价的质控指标之一,具有重要应用前景。

Objective To study the early biological effects of the degranulation of mast cell induced by Shuarlghuanglian injection （SHLI） in vitro, and then to explore the possible quality control in- dex of the satety evaluation on traditional Chinese medicine injections. Methods The mast cell model was RBL-2H3 （ATCC-6562）, SHLI pro＇chased ti＇om Harbin No. 2 Chinese traditional medicine tSctol3~ （Z20043425）. The cell viability was tested by MTT assay and the level of β-hexosaminidase was detected by ELISA method, the morphologic characteristic of mast cells were observed by scanning electron micros- copy, and the concentration of calcium ion in living cells were detected by laser contocal microscopy. Re- sults SHLI of cytotoxic dosage and anaphylactoid reaction dosage induced by SHLI is about ≥ 0.08 rag/ mL, mast cell degranulation reaction may appear less than 1 minute incubated by SHL1 and the peak value of calcium ion concentration in mast cells may appears less than 4 minutes incubated by lower dosage of SHLI. Conclusion SHLI could activate Ca2＋-related signaling pathway in mast cells, induce degranulation reaction and release high level β-hexosaminidase. The results suggest that the Ca：＋ concentration detection method based on RBL-2H3 cells might become one of the quality control indicators tor the satety evaluation of Traditional Chinese Medicine Injection, which has important application prospect.