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。

Objective To explore the effect on degranulation of RBL-2H3 cells and its reasons induced by Shenmai Injection (SMI). Methods Compound 48/80 (C48/80) was used to establish RBL-2H3 cells degranulation model, the release rates of β-hexosaminidase, Tryptase, and histamine and the cell viability of RBL-2H3 cells were detected after treated by different times at the different concentration of C48/80; The indicators and conditions which had a higher degree of release were selected as the preferred study indicators and conditions in the case of that the cell viability was more than 80 percents. RBL-2H3 cells were cultured with the geometric dilution at different concentrations of the stock solution of SMI and its solvent (Tween-80), the morphology of degranulation was observed by neutral red staining. In addition, the releasing rates of β-hexosaminidase and histamine in cell supernatants were detected by chromogenic assay and indirect fluorescence analysis, respectively; The viability of RBL-2H3 cells was tested by cell counting kits. Results The optimal condition of C48/80-induced RBL-2H3 cells degranulation test was 30 min and the best indicators were the release rates of β-hexosaminidase and histamine. When compared with the control group, the release rates of histamine and β-hexosaminidase were no difference and the degranulation by neutral red staining did not appear in SMI groups with the concentrations less than 13.3 g crude drug/L (3 times as much as the clinical concentration) while the release rates of histamine and β-hexosaminidase were significantly increased and the degranulation by neutral red staining appeared in SMI 40 g crude drug/L (9 times as the clinical concentration) group and SMI solvent group that both of them included 1.00 g/L Tween-80. In addition, the results of CCK-8 showed that there was no significant effect in different SMI doses groups on cell viability compared with the control group. Conclusion SMI at the concentration below 3 times as much as the clinical concentration has no significant degranulation of RBL-2H3 cells while at the concentration of 9 times as much as the clinical concentration shows the certain effect on inducing the degranulation of cells which may be related to its solvent that contains Tween-80 and shows no relationship with its toxic effects on the cells. It suggests that SMI is relatively safe at the concentration below 3 times as much as the clinical concentration while it has the risk of allergic reactions at the concentration of 9 times as much as the clinical concentration.

重大新药创制科技重大专项(2013ZX09302303);重大新药创制科技重大专项(2012ZX09301003-001-008);国家自然基金(81001254);河北省科技计划项目(13272503D);北京市科委项目基金(Z131100006513010)