Screening Of LXRα Activity In Water And High-throughput Identification Of Its Effectors

Endocrine disrupting chemicals,a group of exogenous chemicals that can interfere with hormone action in the body,mainly serve as ligands of nuclear receptors to regulate the transcription process of cells,and then affect the endocrine process of the human body,which negatively affects human health and development.A large number of studies have shown that there is a wide range of endocrine disruption phenomena in the environment,in where these substances are commonly detected such as natural water,soil,sediments,and indoor dust.Liver X receptors(LXRs),which are cholesterol-sensitive elements in cells and the body,are members of the nuclear receptor family.LXRs can regulate the occurrence and development of atherosclerotic diseases by regulating the formation of foam cells and the levels of inflammatory factors in the body.As more and more chemicals enter the environment,there are a large number of potential pollutants in the environment that have LXRs activity effects.However,it is difficult to determine the specific identity of the LXRs effect substances that cause such activities.Traditional unknown substance identification technology is difficult to meet the identification of trace active substances in complex environmental matrices due to its shortcomings of relatively independent chemical analysis and biological activity testing,low selectivity,large workload,and poor identification effect,so it is urgent to reform the existing identification Methods for complex pollutants in the environmental matrix.In order to overcome the shortcomings of traditional methods,based on the principle of affinity binding between nuclear receptor proteins and small molecules,this study is using LXRs proteins to specifically capture potential substances,combined with non-targeted analysis and high-throughput identification methods to find potential LXRs effectors in environment.The main results were as follows:(1)In this study,LXRα was taken as an example.First,the p Cold-TF-LXRαrecombinant protein was extracted in the E.coli system.In order to prepare a large number of recombinant proteins,the two conditions of the protein production system(induction temperature and inducer IPTG)were optimized.After further affinity purification by the AKTA protein purification system,p Cold-TF-LXRα recombinant protein with high yield and high concentration was obtained.(2)To verify the method for capturing LXRα effectors based on the principle of affinity binding of nuclear receptor proteins and small molecules,the recovery of standard addition experiments of T0901319 which is the LXRα positive agonist is used to prove the validity and specificity of the method.At the same time,in verifying the practicability of the method,this study found for the first time that in addition to the already reported TPHP and EHDPP,BPADP,TCr P,TNBP,RDP,TEHP,and TDCIPP also have LXRα antagonistic activity,which indicates that the method can be specific capturing effector substances and can be used for high-throughput screening of LXRαeffector substances in environmental samples.(3)In this study,an affinity binding method combined with UHPLC-QE-MS / MS was used to transform the material information in the water samples from the secondary sedimentation tank and effluent of the Gaobeidian Sewage Treatment Plant into chromatographic and mass spectrometric data.These data were analyzed by Progenesis QI and Simca-P.The results of PCA and OPLS-DA showed that there was a significant difference between the experimental group and the control group,indicating that p Cold-TF-LXRα recombinant protein did carry out some substances specific adsorption.Then,155 suspected effector substances were screened out from the secondary sedimentation tank water sample and 113 suspected effector substances were screened from the effluent water sample by means of the fold change of peak intensity,student t test,and manual correction.Finally,compared with the chromatograms and mass spectra of mz Cloud and commercial standards,the structural formulas of two potential effectors were identified,4-Acetamidoantipyrine(Secondary sedimentation tank / Effluent),and Catechol(Secondary sedimentation tank / Effluent).And the yeast two-hybrid experiment was proved that both 4-Acetamidoantipyrine and Catechol have an antagonistic effect on LXRα.(4)Furthermore,this new high-throughput analysis method was used to detect the sample of Drinking Water Plant.The results show that the cardiovascular drug Telmisartan(Influent and the Sand filter)and the triazole fungicide Propiconazole(only in Influent)have the antagonistic effect of LXRα.In summary,four new LXRα antagonists were identified.Although the specific structures of other substances were not identified,the accurate mass,retention time,and so on which can predict the molecular formula of each chemical,could provide important information for the next step of identification.At the same time,this study used the principle of affinity binding to detect the antagonist of LXRα in the water environment for the first time,providing important theoretical experience and technical basis for the subsequent screening of potential pollutants in the environment.