Targeted Metabonomic Study Of Bile Acids In TX Mice By Nuclear Receptor FXR

Objective:Hepatolenticular degeneration(HLD),also known as Wilson’s disease(WD),is a hereditary disorder of copper metabolism.WD patients suffer from impaired copper excretion from the biliary tract,which ultimately leads to the accumulation of copper in various tissues and organs and causes severe damage.Farnesoid X Repoter(FXR)is a nuclear receptor activated by bile acid,which can reduce bile acid accumulation in the liver and avoid hepatotoxicity by regulating bile acid metabolism.FXR can also promote bile acid excretion by regulating bile acid efflux transporter.According to the previous research of our group,intervention of primary bile acid biosynthesis pathway can play a role in the treatment of WD disease.In order to explore the effect of nuclear receptor FXR on bile acid homeostasis in TX mice,targeted metabonomics was used to explore the changes of bile acid composition and content in serum and urine of patients with WD,and to compare the difference of bile acid content in liver and plasma of TX mice(WD transgenic mice)by activating / antagonizing FXR nuclear receptor,and to investigate the effect of FXR nuclear receptor on bile acid spectrum of TX mice,so as to provide basis for the application of FXR in the treatment of WD disease.Methods:The serum and urine were collected from of hospitalized WD patients in the first affiliated Hospital of Anhui University of Chinese Medicine and healthy controls(HC)examined in the physical examination center.LC-MS/MS targeted metabonomics was used to detect the content of bile acids in serum and urine of WD group and HC group,to screen out differential bile acids and to explore the changes of bile acid spectrum in WD patients.In order to explore the effect of bile acid activated nuclear receptor FXR on WD,TX mice,FXR agonist Obeticholic acid(OCA)and FXR antagonist Guggulsterone(GS)were used to observe the morphology of gallbladder in control,control+OCA,model,model+OCA and model+GS group,and to investigate the effect of FXR intervention on gallbladder morphology in TX mice.The hematoxylin-eosin staining(HE staining)method was used to observe the pathological changes of liver tissue of mice in each group,to investigate the degree of liver injury in TX mice and the effect of FXR intervention,to determine the content of total bile acid(TBA)in each group,and to investigate the effect of FXR on cholestasis and TBA level in TX mice.To establish a method for the determination of targeted bile acid metabolites in liver and plasma of TX mice,to determine the contents of bile acids in liver and plasma of each group of mice,to investigate the effect of FXR on bile acid spectrum of TX mice,to screen differential bile acids,and to explore the effect of intervention of nuclear receptor FXR on bile acid disorder in TX mice.Results:1.In clinical study,20 patients with WD and 20 healthy controls were collected.19 kinds of bile acids in human serum and urine were determined by LC-MS/MS,and the contents of bile acids in serum and urine in WD and HC groups were determined respectively.Compared with HC group,the level of serum total bile acid in WD patients was significantly higher,and seven differential bile acids were screened,mainly primary bile acids such as GCDCA,TCDCA,GCA,TCA and CA,while four bile acids in urine of WD patients contributed significantly to the difference between groups,mainly secondary bile acid GUDCA.It is suggested that WD disease is related to bile acid metabolism,and the differential bile acid selected can be used as a marker of WD disease.2.Combined with FXR agonist and FXR antagonist,the effect of FXR intervention on TX mice was investigated.After control administration,there was no significant difference between the control group and the control group.The gallbladder of TX mice in the model group was significantly larger than that in the control group,while the gallbladder of TX mice after OCA intervention was smaller than that of the model group,and the gallbladder was enlarged after GS intervention.HE staining showed that activating FXR could significantly improve the survival rate of hepatocytes and protect the structure and morphology of liver tissue in TX mice.Compared with the control group,the level of TBA in plasma and liver of TX mice in model group increased significantly(P < 0.01),and there was no significant difference between control group and control+OCA group.After administration of OCA,the content of TBA in TX mice decreased significantly(P < 0.05,P < 0.01),while the level of TBA increased significantly after administration of GS(P < 0.05,P < 0.01),indicating cholestasis in plasma and liver of TX mice.Activating FXR could significantly improve the liver function of TX mice and alleviate the liver injury of TX mice by reducing the levels of TBA in liver and plasma,while antagonizing FXR could aggravate the liver function injury continuously.3.The LC-MS/MS methods were established and studied for the determination of bile acid in mice liver and plasma,and the composition of bile acid in liver and plasma of each group was.Compared with the control group mice,the levels of bile acids in the liver and plasma of TX mice were significantly increased,five kinds of differential bile acids were screened in the liver of TX mice consisting mainly CA,TCA and other primary bile acids,and four kinds of differential bile acids were screened in the plasma of TX mice,and the levels of primary bile acids such as TCDCA and TCA were mainly increased.OCA can regulate the bile acid in liver and plasma to normal or close to normal level,while the intervention of GS can further increase the level of primary bile acid.It is suggested that WD disease is related to bile acid metabolism,which increases the ratio of primary bile acid to secondary bile acid,and the activation of FXR can improve the change of bile acid level in TX mice.Conclusion:The disorder of bile acid metabolism is closely related to WD disease.There are obvious bile acid disorders in WD patients and TX mice.There was an increase in the level of total bile acid in WD patients and TX mice,mainly in primary bile acid.FXR is the regulator of bile acid homeostasis.Activating FXR can effectively protect TX mice from liver injury,restore liver function,improve bile acid disorder and adjust bile acid level,while antagonizing FXR can aggravate liver injury and bile acid disorder in TX mice.