Establishment of High-throughput Model for Anaphylactic Reaction in Vitro and Evaluation of Potential Anaphylactogen Isoliensinine
10.3969/j.issn.1005-5304.2016.11.013
- VernacularTitle:体外高通量评价过敏反应模型的建立及其对潜在过敏原异莲心碱的评价研究
- Author:
Li WANG
;
Qing LIU
;
Shifeng WANG
;
Yangyang YU
;
Shengnan SUN
;
Jun JIANG
;
Xinyuan SHI
;
Shiyou LI
- Publication Type:Journal Article
- Keywords:
MrgX2;
evaluation method for anaphylactic reactions;
high throughput;
calcium mobilization assay;
isoliensinine
- From:
Chinese Journal of Information on Traditional Chinese Medicine
2016;23(11):51-56
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a high-throughput evaluation model for anaphylactic reactions; To screen and identify potential anaphylactogens from TCM monomeric compounds.MethodsCell model of stably expressed MrgX2 was established. Recombinate plasmid pmCherry-C1-MrgX2 was transfected to HEK293 to establish cell line for screening model. MrgX2 agonist and antagonist were used to identify the validation and stability of the cell line. A small library consisting of 180 compounds was profiled by using a cell-based calcium mobilization assay to find novel compounds targeting the MrgX2 receptor. EC50 test, IC50 test, specificity validation and cytotoxicity evaluation were carried out to detect the function of the positive agonist.ResultsThe EC50 of C48/80 to MrgX2 model was 2.7 μg/mL and the IC50 of 2-APB (evoked by 10 μg/mL C48/80) was 46.29 μmol/L. The first generation cell model of MrgX2 was similar to the 20th generation, and the Z factor of MrgX2 cell model was 0.78. In the primary screening for agonist, isoliensinine was identified as a novel agonist targeting receptor MrgX2 with an EC50 of 4.5 μmol/L and IC50 of39.47 μmol/L. Moreover, isoliensinine was validated to activate MrgX2 receptor specifically without cytotoxicity. Conclusion A high-throughput evaluation method for anaphylactic reactions can be established in vitro through calcium mobilization assay. A potential anaphylactogen isoliensinine is identified and validated.
