中药注射剂所含吐温-80与过敏反应关系的研究

目的：观察不同浓度吐温-80溶液、吐温-80含量不同的中药注射剂对RBL-2H3细胞脱颗粒的影响,探讨中药注射剂所含吐温-80与过敏反应的关系。方法：体外培养RBL-2H3细胞,加入不同浓度（40、20、10、2、1、0.2、0.1、0.05mg/mL）的吐温-80溶液,之后加中性红染液,计数不同浓度吐温-80溶液各组及对照组的脱颗粒细胞,并计算其百分率,同时检测细胞上清液中β-氨基己糖苷酶及组胺的释放量;测定穿琥宁注射液和香丹注射液中吐温-80的含量,2种中药注射剂对RBL-2H3细胞的半数抑制浓度（IC50）以及加入2种注射液各组细胞释放组胺的量。结果：中性红染色实验显示吐温-80可导致RBL-2H3细胞脱颗粒,表现为肥大细胞体积变大,内有空泡产生;浓度为40、20、10、2、1、0.2、0.1mg/mL的吐温-80溶液各组和RPMI1640对照组导致细胞的脱颗粒百分率分别为（57.38±0.47）、（32.54±2.33）、（21.74±0.72）、（16.96±0.26）、（11.40±1.70）、（9.71±0.26）、（7.22±0.15）和（1.51±1.39）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;浓度为40、20、2、1、0.2mg/mL的吐温-80溶液各组和RPMI1640对照组致细胞β-氨基己糖苷酶的释放率分别为（52.44±1.53）、（18.91±0.77）、（7.50±1.82）、（6.65±0.20）、（6.15±0.27）和（0.35±0.06）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;不同浓度吐温-80溶液引起RBL-2H3细胞释放组胺的量也不同;当吐温-80溶液浓度为20～0.1mg/mL时,RBL-2H3细胞脱颗粒百分率、β-氨基己糖苷酶的释放率及其释放组胺的量均与吐温-80溶液的浓度呈线性关系（r=0.9862,r=0.9849,r=0.9740）。穿琥宁注射液和香丹注射液中吐温-80的含量分别为（0.086±0.004）和（0.070±0.008）mg/mL,2种注射液对RBL-2H3细胞的IC50分别为（57.4±1.2）、（1.0±0.2）μL/mL,穿琥宁注射液和香丹注射液组组胺释放量分别为（2.39±0.01）和（1.87±0.00）ng/mL。结论：吐温-80可引起RBL-2H3细胞脱颗粒释放炎症介质;RBL-2H3细胞组胺的释放量与中药注射剂中吐温-80的含量有关;中药注射剂中所含的吐温-80可能与过敏反应的发生有关。

Study on relationship between Tween-80 contained in injections of Chinese herbal medicine and anaphylactic reactions

Objective： To observe the effects of different concentrations of Tween-80 solutions and injections of Chinese herbal medicine with different contents of Tween-80 on degranulation of RBL-2H3 cells in order to explore the relationship between Tween-80 and anaphylaxis caused by injections of Chinese herbal medicine. Methods： RBL-2H3 cells were cultured in vitro and the different concentrations （ 40,20,10,2,1,0. 2,0. 1,0. 05mg/mL） of Tween-80 solutions were added into the supernatants and then stained with neutral red. The degranulated cells in the different concentrations of Tween-80 solutions and the control groups were counted, respectively; the percentage of degranulated cells were calculated,and the amount of β-hexosaminidase and histamine release from the cells in the supernatants were measured. The contents of Tween-80 in the chuanghuning and xiangdan injections were determined,and the 50% concentration of inhibition （ IC50 ） of the 2 injections to the RBL-2H3 cells and histamine release from the cells in the supernatants in the 2 injection groups were measured. Results： The experiment of neutral red staining showed degranulation of RBL-2H3 cells,manifested as enlarged mast cells with vacuoles. The percentage of degranulation of RBL-2H3 cells in the 40,20,10,2,1, 0. 2,0. 1 mg/mL Tween-80 solution groups and the RPMI 1640 control group were （ 57. 38 ±0. 47） ,（ 32. 54 ±2. 33） ,（ 21. 74 ±0. 72） , （ 16.96 ±0.26） ,（ 11. 40 ± 1. 70） ,（ 9. 71 ± 0. 26） ,（ 7. 22 ± 0. 15） ,and （ 1. 51 ± 1. 39） %,respectively. There were significant differences between the two groups （ P〈0. 05,P〈0. 01） . The percentage of β-hexosaminidase release from RBL-2H3 cells in the 40,20,2,1,0. 2 mg/mL Tween-80 solution groups and the RPMI 1640 control group were （ 52. 44 ± 1. 53） ,（ 18. 91 ± 0. 77） ,（ 7. 50 ± 1. 82） ,（ 6. 65 ± 0. 20） ,（ 6. 15 ± 0. 27） ,and （ 0. 35 ± 0. 06） % ,respectively. There were significant differences between the two groups （ P〈0. 05,P〈0. 01） . The different concentrations of Tween-80 solutions induced different levels of histamine release. There was a linear relationship between the percentage of degranulation,β-hexosaminidase release,histamine release from RBL-2H3 cells and the concentration （ 20-0. 1mg/mL） of Tween-80 solutions （ r = 0. 986 2,r = 0. 984 9,r = 0. 974 0） . The contents of Tween-80 in the chuanghuning and xiangdan injections were （ 0. 086 ± 0. 004） and （ 0. 070 ± 0. 007） μg/mL,respectively. The IC50 of the two injections to RBL-2H3 cells were （ 57. 4 ± 1. 2） and （ 1. 0 ± 0. 2） μL/mL,respectively. The levels of histamine release in the chuanghuning and xiangdan injection groups were （ 2. 39 ± 0. 01） and （ 1. 87 ± 0. 00） ng/mL,respectively. Conclusion： Tween-80 can induce RBL-2H3 cells to release inflammatory mediators; the levels of histamine release from the RBL-2H3 cells are related to the contents of Tween-80 in the injections of Chinese herbal medicine; Tween-80 contained in injections of Chinese herbal medicine may be related to the anaphylaxis.