Study On Biomarkers Of Liver Injury Induced By Realgar Based On Target Metabonomics Of Bile Acid

Objective:Realgar contains arsenic and is one of the 28 toxic traditional Chinese medicines specified by the State Council in the"Administrative Measures on Toxic Drugs for Medical Use".The main component of realgar is arsenic disulfide or tetraarsenic tetrasulfide（As₂S₂ or As₄S₄）,and also contains a small amount of arsenic trioxide（As₂O₃,i AsⅢ,that is,arsenic）.As₂S₂ or As₄S₄ is not easily absorbed in the gastrointestinal tract and is directly excreted from the feces,while i AsⅢis easily absorbed by the gastrointestinal tract and is a toxic component of realgar.The arsenic in realgar is a toxic metal and is recognized as a carcinogen in the world.The accumulation of arsenic is the main reason for the toxic side effects of realgar.A large number of realgar or realgar-containing preparations poisoning incidents are caused by overdose,long-term use and improper processing.Therefore,the safety of the application of realgar and its compound preparations has been controversial.The liver is the main organ for drug metabolism,and it is also most vulnerable to drug toxicity.In recent years,reports of liver damage caused by traditional Chinese medicine have increased.Studies have shown that realgar has obvious hepatotoxicity.Finding the biomarkers of realgar-induced liver injury has important theoretical and practical significance for early effective measures to prevent realgar-induced liver injury and to guide clinical rational use of drugs.Metabolomics technology is currently the main technology widely used in biomarker screening and an important part of systems biology.Bile acid is the main lipid component of bile.It is a general term for a class of cholanoic acid.It can promote the absorption and digestion of lipids and participate in various metabolisms of the body as a signal molecule.The change in quality and quantity is the diagnosis of liver disease.The study of identification and pathogenesis provides valuable information.At the same time,the study of the bile acid metabolism network is of great significance for the study of the mechanism of drug-induced liver injury and the screening and evaluation of drugs for liver protection.Therefore,this study established an ultra high performance liquid chromatography-mass spectrometry（UHPLC-MS/MS）method for the determination of 14 bile acids in mouse liver tissue and plasma.The target metabolomics strategy was used to study the changes in the metabolic characteristics of bile acid profiles in the liver tissue and plasma of mice exposed to 0.15,0.45,and 1.35 g/kg realgar for 8weeks,and to clarify the bile acid biomarkers of realgar-induced liver injury.Methods:1.Establishment of mouse realgar-induced liver injury model:36 male Kunming mice（kunming mice,KM mice）（body weight 20-30 g）were randomly divided into control group and low,medium and high dose realgar dyes according to their body weight Toxic group,9 animals in each group.The control group was given 0.5%carboxy methyl cellulose-Na（CMC-Na）by gavage;The low,medium and high dose realgar group were given 0.15 g/kg,0.45 g/kg and 1.35 g/kg respectively by gavage（0.5%CMC Na as suspension medium）,the liver and whole blood were sacrificed after 8weeks of continuous gavage to prepare plasma.2.Determination of arsenic content:Take about 50 mg of liver tissue or 100μL of whole blood,add concentrated nitric acid for digestion for 24 hours,centrifuge,dilute the supernatant and add ascorbic acid and thiourea to reduce the arsenic content by atomic fluorescence spectrometer.3.Aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,total cholesterol（TC）and total bile acids（TBA）.Determination:Take 100μL of plasma or liver tissue homogenate,and operate according to the kit instructions.4.Observation of liver pathology:hematoxylin-eosin staining（H&E staining）.5.Determination of oxidative damage parameters of liver tissue:Take liver tissue to prepare 10%tissue homogenate,and determine malondialdehyde（MDA）and glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and catalase（CAT）levels.6.Establishment and verification of 14 kinds of bile acid analysis methods in liver tissue and plasma:UHPLC-MS/MS method.7.Determination of genes related to bile acid metabolism in liver tissue:Real-time quantitative polymerase chain reaction（RT-q PCR）was used to detect genes related to bile acid metabolism cholesterol 7α-hydroxylase（Cyp7a1）,bile salt export pump（Bsep）,sodium taurocholate cotransporting polypeptide（Ntcp）,multidrug resistance-associated protein 2（Mrp2）,method The expression level of farnesoid X receptor（Fxr）.8.Bioinformatics:Bile acid concentration data in liver tissue and plasma were analyzed by partial least squares discriminant analysis（PLS-DA）using Metabo Analyst platform to observe the separation of each group.T test（student’s t-test）is used to analyze differences between groups.Bile acids that also meet the variable importance for the projection（VIP）>1 and P<0.05 were selected as candidate markers.The SPSS software was used to analyze the correlation of each sample;the receiver operating characteristic curve（receiver operating characteristic,ROC）was used to evaluate the accuracy of the bile acid biomarkers in diagnosing liver injury caused by realgar.9.Statistical analysis:All data in this study used SPSS 17.0 statistical software for statistical analysis.The experimental data are all expressed as mean±SEM.The unpaired t test and the analysis of variance test were used to compare the expression levels of bile acids in the realgar low,medium,and high dose groups and the control group.When P<0.05,the difference was statistically significant.Results:1.No arsenic was detected in the liver tissue and whole blood of mice in the control group.The liver arsenic and blood arsenic content of mice in the low,medium and high dose groups were significantly higher than those of the control group,with significant differences（P<0.01）.2.The body weight of mice in each group gradually increased with the feeding time,and the liver organ coefficient showed a downward trend.The ALT activity in the plasma of mice in the realgar-exposed group showed a rising trend,the AST activity in the plasma showed a trend of first decreasing and then increasing,and the concentration of TC and TBA in the plasma showed an increasing trend.The results of pathological studies show that realgar exposure can cause significant changes in liver tissue morphology in mice.3.Compared with the control group,the MDA content and SOD activity in the liver tissue of the realgar-exposed mice were significantly increased（P<0.05）,and the GSH-Px activity was significantly reduced（P<0.05）.4.Established a UHPLC-MS/MS analytical method for the determination of 14 bile acids in mouse liver tissue and plasma,and used it for the determination of bile acids in liver tissue and plasma of mice in the control group and realgar-exposed group.Eleven bile acids were detected in mouse liver tissues,and 12 bile acids were detected in mouse plasma.5.The percentages of the three types of bile acids in mouse plasma and liver tissues are all taurine-bound bile acids>free type>glycine-bound type.After realgar exposure,the proportion of free bile acids in the liver tissues of mice showed an increasing trend,and the proportion of taurine-bound bile acids appeared to decrease;in contrast to liver tissues,the proportion of taurine-bound bile acids in mouse plasma appeared after realgar exposure With the increasing trend,the proportion of free bile acids has a decreasing trend,and the proportion of glycine-bound bile acids has also shown a decreasing trend.6.Compared with the control group,the ratio between free bile acid and glycine-bound bile acid,and the ratio between free bile acid and taurine-bound bile acid in the plasma of mice in the realgar-exposed group increased;In liver tissues,the ratio of free bile acids to taurine-bound bile acids decreases,and the ratio of primary bile acids to secondary bile acids decreases.8.Compared with the control group,chenodeoxycholic acid（CDCA）,hyodeoxycholic acid（HDCA）,taurochenodeoxycholic acid（TCDCA）and ursodeoxycholic acid Ursodeoxycholic acid（UDCA）was significantly increased in the liver tissue of mice in the realgar-exposed group（P<0.05）;glycodeoxycholic acid（GDCA）was significantly decreased in the liver tissue of mice in the realgar-exposed group（P<0.05）.Deoxycholic acid（DCA）,taurocholic acid（taurocholic acid,TCA）,tauroursodeoxycholic acid（TUDCA）,TCDCA increased significantly in the plasma of mice exposed to high-dose realgar（P<0.05）.Realgar exposure affects the correlation between bile acids in plasma and liver tissues of mice.The correlation between taurine-bound bile acids and free bile acids is good and positive,and there is a negative correlation between glycine-bound bile acids.9.Correlation analysis between bile acids and oxidative damage indicators found that TCDCA and SOD in the plasma of realgar-infected mice were positively correlated,and TCDCA and GSH-Px were negatively correlated in liver tissue.As the dose of realgar increased,the correlation between glycine-binding bile acids in plasma and oxidative damage indicators increased.10.Using PLS-DA to analyze the bile acid metabolism profile of mice,the results found that the separation of the plasma bile acid metabolism profile of the realgar-exposed group and the control group was more obvious than that of the liver bile acid metabolism profile.OPLS-DA,VIP and P values were used for biomarker screening,and it was found that TCA and TCDCA in plasma and HDCA and TCDCA in liver tissue can be used as potential biomarkers for realgar-induced liver injury.11.ROC curve results show that the AUC of HDCA and TCDCA in liver tissue is less than 0.3.The AUC of TCA and TCDCA in plasma is less than 0.1.12.Compared with the control group,the expressions of CYP7A1,FXR,BSEP and MRP2 m RNA in the liver tissue of mice exposed to realgar were increased,while NTCP m RNA was significantly decreased.Conclusion:1.The arsenic in realgar can accumulate in the liver tissue of mice and cause morphological changes,abnormal liver function,and oxidative damage of liver tissue in mice.2.Realgar can cause changes in the bile acid profile of mice,resulting in abnormal bile acid metabolism.3.TCDCA is a sensitive bile acid marker of realgar induced liver injury in mice...