Application Of Mrgprb2 Gene Knockout Mouse Model In Anaphylactoid Reaction Induced By Houttuynia Cordata Injection And Construction Of Mrgprx2 Humanized Mouse Model

Houttuynia cordata injection is prepared from traditional medicine Houttuynia cordata by modern methods.Because of its various pharmacological activities,it is widely used as antipyretic,detoxification,anti-inflammatory and other drugs.However,with the increasing clinical application of Houttuynia cordata injection,reports of adverse events about it are also increasing.Anaphylactoid reaction is one of the main adverse reactions of Houttuynia cordata injection,and MRGPRX2 is one of important gene mediating anaphylactoid reaction.,and MRGPRX2 is an important gene that mediates anaphylactoid reactions.The research group has successfully constructed a Mrgprb2 gene knockout mouse model,Mrgprb2 is a mouse homologous gene of MRGPRX2,and used this model mouse and wild-type mouse to evaluate the anaphylactoid reaction induced by Houttuynia cordata injection in vitro and in vivo according to various indicators(including β-hexosaminidase release,histamine release,electron microscopy,and Evans blue staining).In this research,RNA-seq high-throughput sequencing technology was utilized,which included Mrgprb2 knockout model mice and wild-type mice,to assess and authenticate the anaphylactoid reaction induced by Houttuynia cordata injection.Whether 2-Undecanone and Tween 80 can cause anaphylactoid reaction was evaluated in vivo and in vitro,and Houttuynia cordata injection and C48/80 were selected as control drugs to determine the anaphylactoid components in Houttuynia cordata injection.Finally,a MRGPRX2 gene humanized mouse model was constructed and identified by DNA and sequencing.Here are the experimental results:(1)In this study,MRGPRX2 gene humanized mouse model was constructed.A total of two vector injections were carried out.After DNA and sequencing detection,A total of 19 homozygous positive F0 generation mice were obtained.However,the mice are in poor physical condition and there were 4 mice remaining and ovarian transplants were carried out.No mice were produced in the breeding process of mice receiving ovarian transplantation.(2)By analyzing the transcriptome of mast cells in gene knockout mice and wildtype mice injected with PBS and Houttuynia cordata injection,it was found that the m RNA library was successfully constructed.In wild-type mice,Houttuynia cordata injection mainly mediates anaphylactoid reactions through Glutathione metabolism,Pyrimidine metabolism,Aflatoxin biosynthesis,Prolactin signaling pathway,Longevity regulating pathway – worm,Circadian rhythm,Mineral absorption,Signaling pathways regulating pluripotency of stem cells,Alanine,aspartate and glutamate metabolism,FoxO signaling pathway.After knocking out Mrgprb2,the anaphylactoid reaction was significantly reduced,mainly involving P53 signaling pathway,PPAR signaling pathway,retinol metabolism,indole alkaloid biosynthesis,cell cycle-Caulobacter,FoxO signaling pathway,Betalain biosynthesis,circadian rhythm-fly,TGF-β signaling pathway,isoquinoline alkaloid biosynthesis,etc.Based on KEGG enrichment results,five genes were selected for q PCR verification in each group.The results were in agreement with the transcriptome sequencing results.(3)According to the release rate of β-hexosaminidase,the release amount of histamine and the degree of Evans blue staining,the sensitization of methyl nonanone and Tween 80 was evaluated.Before knocking out Mrgprb2,the indexes of Tween 80 group were significantly improved,which was significantly higher than that of Houttuynia cordata injection and 2-Undecanone group.The indexes of 2-Undecanone group increased slightly,which was lower than that of Houttuynia cordata injection and Tween 80 group.After knocking out Mrgprb2,the three indexes of each group were lower than those of wild type,especially in Tween 80 group,and the decrease of2-Undecanone group was small.The β-hexosaminidase released by mouse mast cells in the 2-Undecanone group was little higher than that of the blank group,yet the remaining data were comparable to the blank group.Based on the above experimental results,2-Undecanone was not detected as a sensitizer;Tween 80,an important excipient in Houttuynia cordata injection,can bind to MRGPRB2 receptors to stimulate mast cells and cause anaphylactoid reactions.