Based On Metabolomics And Network Toxicology, The Molecular Mechanism Of Epimedium-induced Liver Injury Was Preliminarily Explored

Background:Epimedium is a traditional Chinese medicine for tonifying kidney,strengthening muscles and bones,dispelling dampness,which also have anti-inflammatory,anti-tumor,promoting hematopoietic function and bone metabolism and other pharmacological activities.Epimedium has a wide range of clinical application,is often used in the treatment of male infertility,osteoporosis,hypertension and coronary heart disease.However,in recent years,there have been numerous reports on the adverse reactions of Epimedium and its proprietary Chinese medicine.Epimedium has changed from a widely recognized "non-toxic" traditional Chinese medicine to a "potentially toxic" traditional Chinese medicine.The most common side effects of Epimedium are liver injury and the consequences are also the most serious.However,there are few studies on the hepatotoxicity of Epimedium,and the mechanism of hepatotoxicity of Epimedium is urgently needed to be explored.The study on the mechanism of hepatotoxicity of traditional Chinese medicine combining metabonomics and network toxicology is a new idea for the research on the control of toxicity of traditional Chinese medicine.Research objectives:In this study,the potential toxic components of Epimedium were screened out through network toxicological study,and the molecular mechanism of Epimedium induced liver injury was preliminary explored by metabonomics method,and the pharmacokinetic characteristics of Epimedium and Fructus Ligestii were investigated to explore the possibility of toxicity reduction in combination of Epimedium and Fructus Ligestii,so as to provide important scientific basis for clinical rational drug use of Epimedium.Methods and results:(1)The chemical constituents of Epimedium were identified by UPLC-QE-Orbitrap-MS,and 62 compounds were obtained,including 57 flavonoids,4 organic acids and 1 alkaloid.Then,combined with the theory of "plasma pharmacology",the chemical components in the plasma of Epimedium after administration were analyzed,and 17 kinds of migratory components in blood were identified.The main constituents were flavonoids,and most of them were dehydrated Icaritin as parent nucleus.Therefore,it is speculated that Icaritin may be closely related to liver injury caused by Epimedium.(2)The toxicity network of "Epimedium-component-target-liver inj ury" was constructed by using network toxicology research methods,and then the key hepatotoxic component Icaritin was screened.Further study on this compound revealed that its toxicity mechanism may be closely related to the regulation of oxidative stress,proliferation and apoptosis and other physiological processes,which confirmed the previous hypothesis.(3)Human normal liver cell(L-02)model was selected to verify the hepatocytotoxicity of Icaritin by MTT method.Then,non-targeted metabonomics technique was used to investigate the changes of metabolic profile of cells in different durations of Icaritin administration compared with the control group,and 106 different metabolites were obtained.A total of 14 potential biomarkers significantly associated with cell survival were screened by Pearson correlation analysis combined with L-02 cell survival rate.Through the pathway enrichment analysis of all the different metabolites,it was found that the changes of cell metabolism were mainly involved in glutathione metabolism,arginine and proline metabolism,glycerolipid metabolism,phenylalanine metabolism,pentose and glucuronic ester mutual conversion,which preliminarily revealed the mechanism of hepatotoxicity induced by Epimedium.(4)Based on UPLC-QQQ-MS,a method was established for the determination of icariin,epimedin A,epimedin B,epimedin C,baohuoside I and specnuezhenide in biological samples,and the method was successfully applied to study the pharmacokinetics after the combination of Epimedium and Fructus Ligestii.The results showed that icariin,epimedin A,epimedin B,epimedin C,baohuoside I could be rapidly absorbed into blood after intragastric administration,and the second peak time of the drug in vivo was earlier after the combination of Epimedium and Fructus Ligestii,indicating that the metabolic rate may be changed.In addition,the combination with Fructus Ligestii could significantly reduce the concentration of 5 flavonoids in Epimedium in blood and increase their elimination rate in vivo.Conclusions:This study focused on the "potential hepatotoxicity" of Epimedium,aiming to identify its potential toxic components and reveal its toxic mechanism.Finally,Icaritin was determined to have certain hepatocytotoxicity,and its toxicity mechanism may be related to glutathione metabolism.A total of 14 potential biomarkers were screened out.In addition,this study also found that the combination of Epimedium and Fructus Ligestii could significantly inhibit the enrichment of five flavonoids in Epimedium,which may have attenuation of virulence,in order to provide a reference for clinical rational drug use of Epimedium.