Establishment Of In Vitro High - Throughput Evaluation Of Allergic Reaction Model And Screening And Validation Of Potential Allergens In Traditional Chinese Medicine Compounds

It is reported that allergic reactions induced by Traditional Chinese Medicine (TC M), especially Chinese herb injections, happens from time to time. This has been the main factor restricting the development of TCM, and therefore, it is important to i mprovethe level of TCM quality control to improve the clinical medication safety. At present, the method to evaluate allergic reactions is mainly divided into vivo method a nd vitro method. Vivo method mainly refers to animal experiments and it has mamy disadvatages, includeing long experimental period, time-consuming work, subjectivity a nd poor accuracy. Thus, vivo method has been unable to meet the modern needs of h igh-throughput screening (HTS). Vitro method mainly refers to immune model and cell model, however, immune model has the disadvantages including higher false positive and false negative rate. Currently, it is reported that some substance could induce mas t cell to degradulate and release histamine. Moreover, D. McNeil Benjamin pointed ou t that MrgX2 is an important drug target inducing allergic reactions.Thus,, we established a recombinant cell model stably over expressing receptor M rgX2, which can be used for the evaluation of the sensitive substances. Based on cell model, a screening model based on calcium flow analysis was established and the ev aluation system based on RTCA platform was preliminarily exploredMoreover, the two methods were compared. We used calcium current high-throughput screening model b ased on HEK293/MrgX2 cell lineto evaluate allergenicity of 180 monomer compounds.. The study was processed to provide a more sensitive, rapid and effective method for quality control of TCM, especially Chinese herb injections. The study was also aime d to provide reference for potential allergenicity and to minimize allergic events. The main results are as follows:Part 1:Establishment of recombinant cell lines over-expressing MrgX2 receptorFirstly, we constructed the eukaryotic plasmid vector pmCherry-C1 with a red flu orescent tags, and then the target gene fragment was inserted into vector plasmid to c onstitute the recombinant plasmid pmCherry-Cl-MrgX2. Before transfection, we should test the quality and purity of plasmid via electrophoresis and UV detection and the r esults showed that quality and purity of recombinant plasmid with MrgX2 was able to meet the needs of transfection. Then, the plasmid transfected HEK293 host cell via s table transfection method. After antibiotic G418 screening,32 successfully transfected cell lines was picked up, and therefore,5 successfully overexpressing MrgX2 monoclo nal cell lines were obtained. Finally, we picked the cell line with good condition, high stability and sensitivity to take futhur study.Part 2:establishment of evaluation model based on calcium flow and RTCA anal ysis methodFirstly, we examined the functionality and stability of HEK293/MrgX2 using C4 8/80 as positive agonist and 2-APB as positive antagonist. EC50 and IC50 were tes ted using calcium flow detection method and EC50 was consistent with the reported r eference.. With the range from P0 to P30, we measured EC50 once every five generat ions and the results show that the stability of the cell line is sufficient to meet the n eeds of the screening system. In order to verify the stability and reliability of calcium screening system, we apply the Z factor to evaluate it and the results (Z=0.77) sh owed that this calcium flow screening model meet the needs of HTS.As to explore of RTCA modle, we still use C48/80 and 2-APB as positive agoni st and antagonist respectivly and we also tested the EC50, IC50, Z factor. The results showed that C48/80 and 2-APB on the MrgX2 showed dose-dependent manner. To test the stability and reliability of the system, we evaluated Z factor and result indie ated that the evaluation system is sufficiently stable and reliable. RBL-2H3 endogenou sly expresses MrgX2 homologous gene MRGPRB2, thus, we tested the C48/80 on t he RBL-2H3 and its EC50 was consistent with reference report.Thus, we initially esta blished evaluation system based on RTCA analysis method. We then compared the tw o methods by comparing the EC50, IC50, Z factor and the results show that we can c ombine the two approaches to evaluate and validate allergens.Part 3:Application of two evaluation models lines over-expressing MrgX2 receptoBased on calcium flow HTS evaluation model, we evaluated 180 Chinese medicine compound and found four unreported MrgX2 agonists, sinomenine, liensinine, isoliensi nine and neferine. There EC50 values in the calcium flow filter system were 1.997 um ol·L-1、8.924 μmol·L-1.10.31 μmol·L-1 10.29 μmol·L-1. IC50 of positive antagonist 2-APB were 31.55 μmol·L-1、37.76 μmol·L-1、22.11 μmol·L-1、68.94 μmol·L-1. To verify whether this four positive compound is MrgX2 specific agonist, firstly, we tested ago nistic effect on HEK293 host cells, then, we tested them for other six sensory-related receptor, TRPA1, TPRV1, TRPV2, TRPV3, TAS2R and ETB receptors and results sho wed that they can not be activated by HEK293 endogenous receptors and other 6 rece ptor, and therefore, they are specific MrgX2 agonist. In order to observe toxicity of MrgX2 agoniststs, we apply ATP Glo fluorescent method to evaluate them. The results suggested that all the positive compounds showed no cytotoxicity. Furthermore, this s tudy used real-time label-free detection to verify the results.