Establishment of in vitro evaluation model of pseudoallergy and screening of its indexes

LI Wei; CHEN Meng; HUANG Zhi-ying; JIANG Hua; LI Bo

Abstract：Objective:To compare the sensitivity of three kinds of cell lines (RBL-2H3,P8l5 and Ku812 cells) and their markers in the evaluation of drug pseudoallergy and explore the applicability of the in vitro models for the evaluation of pseudoallergy.Methods:C48/80 was used as the positive drug.The cells were treated with different concentrations of C48/80 for 30 and 60 min,respectively.Cell degranulation was evaluated by detecting the release rate of β-hexosaminidase,the ratio of Annexin V positive cells,and the release amounts of IL-4 and TNF-alpha.Then,the applicability of the models was studied though testing the reported anaphylactoid reaction drug Tween 80.At the same time,the pseudoallergy induced by different concentrations of Tween 80 was evaluated.Results:After treatment with C48/80,the release rate of β-hexosaminidase,the ratio of Annexin V positive cells,and the release amounts of IL-4 and TNF-alpha in three kinds of cells were all significantly increased in a dosedependent manner.However,except for the RBL-2H3 cells,the amount of IL-4 released in the other two cell lines was very small,and the amount of TNF-a detected in all three kinds of cell lines was very small.The rate of Annexin V positive cells increased significantly compared with the other three indicators.In the same stimulating conditions,the percentage of Annexin V positive cells and the release rates of 3-hexosaminidase in P815 and Ku812 cells increased significantly earlier than that of RBL-2H3 cells,and the burst effect was more obvious.The results of Tween 80 administration were basically identical with that of C48/80 administration.In addition,the study also found that when the concentration of Tween 80 was greater than 0.05 mg· mL-1,there was a risk of causing allergic reaction.Conclusion:β-hexosaminidase release rate and AnnexinV positive cell rate are two stable,reliable and reproducible indexes for the evaluation of pseudoallergy.IL-4 and TNF-alpha release are more suitable as auxiliary detection indicators of pseudoallergy.The Annexin V positive cell rate may be more sensitive and early to detect the occurrence of pseudoallergy.The cell degranulation of P815 and Ku812 cells occurred faster and more severe than that of RBL-2H3 cells under the same stimulating conditions.These data together suggest that P815 and ku812 cells might be utilized as potential early,fast and sensitive in vitro models for screening suspicious allergens and early screening of drug-induced pseudoallergy.