Study On Liver Injury And Metabolic Pathway Of Aurantio-obtusin Based On Omics

Recently,there have been more and more reports on the toxic and side effects of natural anthraquinone compounds,such as emodin,rhein,chrysophanol,aloe-emodin,etc.,as natural anthraquinone derivatives,which are widely present in traditional Chinese medicines such as Rhubarb,Polygonum multiflorum,Cassiae semen and other traditional Chinese medicines.At present,the hepatotoxic factors of Polygonum multiflorum and Rhubarb are mainly attributed to these anthraquinones.Although anthraquinones have a wide range of pharmacological effects,they also have potential hepato-nephrotoxicity and other toxic and side effects,especially in high dose and long-term use.Therefore,there are certain safety risks in clinical use of these drugs.As a unique anthraquinone active component of Cassiae semen,aurantio-obtusin has many biological characteristics,such as anti-allergy,anti-inflammation,anti-hyperlipidemia,anti-osteoporosis,anti-hypertension,etc.Therefore,this project focused on the safety of aurantio-obtusin,which is divided into the following five parts.Firstly,this study explored the hepatotoxicity of aurantio-obtusin on male Sprague Dawley rats.The animals were randomly divided into blank control group,and different doses of aurantio-obtusin group:low dose(4 mg/kg),medium dose(40mg/kg)and high dose(200 mg/kg).Each group was given aurantio-obtusin for 28days,and the control group was given the same amount of 0.5%CMC-Na aqueous solution.Subsequently,serum biochemical,hematological and pathological examinations were performed,and liver organ coefficients were measured.Serum metabolomics was used to identify biomarkers that may be related to hepatotoxicity.Compared with the blank control group,there was no abnormality in the low-dose group.The middle and high-dose groups had obvious liver injury.At the same time,the levels of alanine aminotransferase,aspartate aminotransferase and alkaline phosphatase increased.Serum metabolomics analysis showed that aurantio-obtusin induced 28 endogenous markers related to liver injury,which were mediated by bile acid,fatty acid,amino acid and energy metabolism.Secondly,based on the results of the first part of serum metabolomics,the hepatotoxicity of aurantio-obtusin was dose-dependent.After the last administration,24-hour urine of rats was collected and analyzed by ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-QTOF/MS).Twenty-three metabolites were identified as potential biomarkers,of which 10 were up-regulated,including xanthine,hippuric acid,5-L-glutamine-taurine,etc.,and the other 13 were down regulated,including thymidine,3-methyldioxygindole,cholic acid,etc.These significant biomarkers indicate that purine metabolism,taurine and hypotaurine metabolism,primary bile acid biosynthesis,pyrimidine metabolism,and tryptophan metabolism play an important role in the hepatotoxicity of aurantio-obtusin in rats.Thirdly,in order to further understand the differential protein expression related to the hepatotoxicity mechanism of aurantio-obtusin,based on the previous results,the hepatotoxicity of aurantio-obtusin was dose-dependent,so the liver of rats in blank control group and high-dose aurantio-obtusin group was selected for non-target analysis quantitative proteomics research.High dose of aurantio-obtusin significantly inhibited the activities of CYPs,SULTs and other enzymes,and induced the activities of UGTs,GSTs and other enzymes,involving drug metabolism,inflammatory factor regulation,bile secretion,fatty acid metabolism and other pathways,which provided the basis for the metabolism of aurantio-obtusin in vivo and the disorder of endogenous metabolites.Fourthly,based on the results of the first and second parts,some of the endogenous metabolites identified by metabolomics are related to the metabolism of gut microbiota,and the microbial modification regulated by bile acid is an important mechanism of the interaction between microorganisms and host,which not only affects liver diseases,but also affects other organs and metabolic pathways.The gut microbiota of mice with liver injury induced by aurantio-obtusin was studied by high-throughput 16S r RNA sequencing.The relative abundance of Bacteroidetes decreased gradually,while the relative abundance of Firmicutes,Proteobacteria,Tenerictes,Actinobacteria and Verrucomicrobia increased gradually.This may be one of the mechanisms of hepatotoxicity.Fifthly,based on the results of the third part of proteomics,aurantio-obtusin can significantly affect the process of drug metabolism.The purpose of this study is to establish a method to study the metabolic process of aurantio-obtusin in normal rats and liver injured rats.By using ultra performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)and Metabolynx^TM software,the metabolic types of aurantio-obtusin(4 and 200 mg/kg)in rats after 28 days of intragastric administration were investigated.A total of 39metabolites were detected,three of which were compared with the reference materials.In normal rats,the main metabolic pathways of aurantio-obtusin are glucuronization and sulfation,while in rats with liver injury,demethylation,dehydroxylation and reduction are also observed,which is considered to be the unique metabolic pathway and metabolites in the model of liver injury induced by aurantio-obtusin.Aurantio-obtusin induces hepatotoxicity in a dose-dependent manner and mediates the metabolism of drugs and endogenous biomarkers by changing metabolic enzymes and gut microbiota.In particular,the changes of bile acid metabolism and drug metabolic enzymes during administration deserve more attention.In this paper,the toxicity of aurantio-obtusin was studied by combining metabolomics,proteomics and gut microbiota for the first time.In addition,this study is helpful to elucidate the metabolic characteristics of other anthraquinones.