Development Of Chemokine Receptor CCR3 Screening Model And Discovery Of Its Natural Antagonist

Atopic dermatitis（AD）is a chronic,relapsing inflammatory skin disease.In recent years,the incidence of AD has increased year by year,which has greatly affected people’s quality of life.However,most AD is still incurable,and glucocorticoids or immunosuppressants with large adverse reactions are still used in clinical treatment.In recent years,biologics such as Dupixent and small-molecule JAK inhibitors such as Tofacitinib and Upadacitinib have also been used in systematic treatment of AD,improving the efficacy of AD,but there are also problems such as high price and serious adverse reactions.Therefore,it is of great significance to carry out research on AD targeting drugs.Chemokine receptors are a class of seven transmembrane G-protein-coupled receptors（GPCR）,which can mediate chemokines to recruit various white blood cells to target sites to perform their functions.Chemokines and their receptors play an important role in mediating cell migration and resisting pathogen invasion,and are closely related to the occurrence and development of inflammation.Chemokine-chemokine receptor axis is considered a very promising drug target for the treatment of inflammatory and immune diseases.Chemokine receptor 3（CCR3）,a member of the chemokine receptor family,is selectively expressed in eosinophils,basophils,and a part of TH2 cells.By binding ligands such as CCL26 chemokines,chemokines of inflammatory cells and tissue infiltration can trigger allergic inflammatory diseases,such as AD.Therefore,antagonizing CCR3 receptor may be an effective treatment strategy for AD.Traditional Chinese medicine prescriptions have a long history and are composed of a variety of medicinal materials,which contain a lot of chemicals.More than 140formulations have been approved by pharmacopeia as ethical drugs,and are widely used in clinical treatment of a variety of diseases.Some classical prescriptions,such as Liuye and Zhudock decoction,Xiaofeng Powder,Nepeta and forsythia decoction,and Sanmiao Powder,have been proven to have good efficacy in treating AD.Therefore,these clinically effective Chinese medicines may be an important source for the discovery of natural CCR3 antagonists with therapeutic effects of AD.Objective:Antagonism of CCR3 receptor may be an effective means to treat atopic dermatitis or other related allergic diseases,so it is of great value to find novel CCR3 receptor antagonists.Natural products are an important source of drug research and development.Some traditional Chinese medicines are considered to have good clinical efficacy in the treatment of AD,but their active substances and action targets are unclear.Therefore,we hope to discover natural CCR3 receptor antagonist drugs based on clinically effective Chinese medicines for AD,and establish AD disease models in vitro to study the mechanism of active drugs.Method:First,we independently constructed stably transfected CHO-K1-Gα15-CCR3 cell lines,and then constructed a high-throughput screening model for CCR3 ligand and analyzed the pathway using labelled free cell integrated pharmacology（CLIP）technology,and verified the activity of potential virtual screened CCR3 antagonists using this model.In addition,we constructed an in vitro inflammatory model of human immortalized epidermal HaCaT cells induced by inflammatory factors.Results:We successfully established CHO-K1-Gα15-CCR3 stable cell line,which was confirmed by gene expression level and confocal fluorescence imaging,and successfully established CCR3 antagonist screening model with this cell.Through pathway analysis experiments,it was found that activation of CCR3 stimulated Gq-PLC-Ca2+and MAPK-P38 pathways.The computer virtual screening technology was used to score and rank 266compounds of 15 kinds of commonly used dermatological Chinese medicines binding to CCR3 receptor,and the activity of the top 26 compounds was verified.Finally,a new CCR3 antagonist emodin was identified.The IC50 of CCR3 antagonistic activity was27.39±0.0017μM.In addition,we successfully constructed the AD inflammation model of HaCaT cells in vitro.Chemokines CCL7 and TLSP,key indicators of inflammation related to AD,were significantly increased after the stimulation of inflammatory factor TNF-α,and the expression level of barrier protein LOR was also significantly up-regulated.Conclusion:1.This study provides a different screening technique for active CCR3 compounds than traditional ones.2.Activation of CCR3 receptors leads to activation of Gq-PLC-Ca²⁺and MAPK-P38 pathways.3.Emodin has obvious inhibitory effect on CCR3 target.4.Emodin may have therapeutic effects on AD,but its efficacy and mechanism of action need further study.