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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。
OBJECTIVE: To observe the effects of different concentrations of Tween-80 and Tween-80 on the degranulation of RBL-2H3 cells and to explore the relationship between Tween-80 and allergic reactions in traditional Chinese medicine injection. Methods: RBL-2H3 cells were cultured in vitro. Tween-80 solution with different concentrations (40,20,10,2,1,0.2,0.1,0.05mg / mL) was added. After adding neutral red dye, Tween -80 solution of each group and the control group of degranulation cells, and calculate the percentage, while the detection of cell supernatant β-hexosaminidase and histamine release; Determination of Chuanhuning injection and Xiangdan injection spit Temperature -80 content, half-inhibitory concentration (IC50) of two kinds of Chinese medicine injection on RBL-2H3 cells and the amount of histamine released by each group of cells added with two kinds of injection. Results: The results of neutral red staining showed that Tween-80 could lead to the degranulation of RBL-2H3 cells, showing the enlargement of mast cells and the generation of vacuoles. The concentrations of 40, 20, 10, 2, 1, 0.2 and 0.1 (57.38 ± 0.47), (32.54 ± 2.33), (21.74 ± 0.72), (16.96 ± 0.26), (11.40 ± 0.26), respectively in each group of Tween-80 solution with mg / mL and RPMI1640 control group 1.70), (9.71 ± 0.26), (7.22 ± 0.15) and (1.51 ± 1.39)% respectively. There was significant difference between the two groups (P <0.05, P <0.01) , 0.2mg / mL Tween-80 solution and RPMI1640 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 was a significant difference between the two groups (P <0.05, P <0.01) Amounts of amines were also different. When the concentration of Tween-80 solution was 20 ~ 0.1mg / mL, the percentage of degranulation of RBL-2H3, the release rate of β-hexosaminidase and the amount of histamine released were similar to those of Tween -80 solution was linear (r = 0.9862, r = 0.9849, r = 0.9740). The contents of Tween-80 in Chuanhuning injection and Xiangdan injection were (0.086 ± 0.004) and (0.070 ± 0.008) mg / mL, respectively. The IC50 values of the two injection solutions on RBL-2H3 cells were (57.4 ± 1.2) (1.0 ± 0.2) μL / mL. The release rates of histamine in Chuanhuning injection and Xiangdan injection group were (2.39 ± 0.01) and (1.87 ± 0.00) ng / mL, respectively. Conclusion: Tween-80 can cause the degranulation of RBL-2H3 cells to release inflammatory mediators. The amount of histamine released from RBL-2H3 cells is related to the content of Tween-80 in Chinese traditional medicine injection. Tween-80 And the occurrence of allergic reactions.