The Role Of Farnesoid X Receptor In Oleanolic Acid-induced Cholestatic Liver Injury In Mice

Objective:To explore the role of farnesoid X receptor（FXR）in oleanolic acid-induced cholestatic liver injury in mice.Methods:Male wild type mice（WT）and male FXR knockout mice(FXR^-/-)were randomly divided into control group（corn oil,C）,oleanolic acid low dose group（457mg/kg,L）and high dose group（685.5 mg/kg,H）.Mice were intragastric administered with oleanolic acid once a day.Mice were sacrificed after 3 hours of oleanolic acid single dose administration,as well as after oleanolic acid administration for 4 days ended 3 hours and24 hours.Biochemical kits were used to detect serum biochemical indicators;HE staining method was used to observe the morphology changes of liver tissue;UPLC-MS technology was applied to analyze changes of bile acid components in liver tissue,serum,and bile;RT-PCR was used to detect the level of bile acid-related genes and Western blot method was used to detect expression of BSEP membrane protein.Results:1.Oleanolic acid continuous administration for 4 days,24 hours after the last dose:In WT mice,compared with the control group,ALT,AST,ALP,and TBA were increased in oleanolic acid high dose group,histopathology showed extensive necrosis of hepatocytes.In FXR^-/-mice,compared with the control group,ALT,AST,ALP,and TBA were increased in oleanolic acid high dose group,and histopathology indicated hepatocyte swelling.In oleanolic acid high dose group,compared with WT mice,ALT,AST,and ALP in FXR^-/-mice were reduced,suggesting that liver damage of FXR^-/-mice was smaller than that of WT mice.UPLC-MS results showed that bile acids in the liver,serum and bile of WT mice and FXR^-/-mice were mainly conjugated bile acids after oleanolic acid administration.Compared with the control group,in WT mice,conjugated bile acids（TCA,TUDCA,TCDCA,T-βMCA）in serum in oleanolic acid high dose group were increased,conjugated bile acids（TCA,TDCA,TCDCA）in the bile were decreased,conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）in the liver were increased;In FXR^-/-mice,compared with the control group,conjugated bile acids（TCA,TUDCA,TCDCA,T-βMCA）in serum in oleanolic acid high dose group were increased,conjugated bile acids（TCA,TDCA,TDCCA）were reduced in the bile,conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）in the liver were increased;In oleanolic acid high dose group,compared with WT mice,serum conjugated bile acids（TCA,TUDCA,TCDCA,T-βMCA）in FXR^-/-mice were reduced,conjugated bile acids（TCA,TDCA,TCDCA）in the bile were increased,and conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）in the liver were increased.The results of bile acid-related genes and Western blot showed that in WT mice,compared with the control group,the expression of Fxr mRNA in oleanolic acid high dose group was down-regulated,bile acid efflux transporters Bsep,Mdr2,Mrp2 mRNA were down-regulated,and OstβmRNA expression was up-regulated.Detoxification enzymes Cyp3a11,Ephx1,Ugt2b5 and conjugated enzymes Bacs,Baat mRNA were down-regulated,the expression of BSEP protein was down-regulated;Fxr mRNA was not basically expressed in FXR^-/-mice,and in FXR^-/-mice,compared with the control group,Bsep,Mdr2,Mrp2,Bacs,Baat mRNA were down-regulated in oleanolic acid high dose group,detoxification enzymes Cyp2b10,Ephx1,Ugt1a1 mRNA were up-regulated,the expression of BSEP protein was down-regulated;In oleanolic acid high dose group,compared with WT mice,the expression of Bsep,Bacs,Baat,Cyp3a11,Cyp2b10,Ephx1,Ugt1a1,Ugt2b5 mRNA and BSEP protein in FXR^-/-mice increased.2.Oleanolic acid continuous administration for 4 days,3 hours after the last dose:In WT mice,compared with the control group,ALT in oleanolic acid high dose group was increased,AST,ALP and TBA showed an increasing trend,liver histopathology showed focal necrosis and inflammatory cell infiltration.In FXR^-/-mice,compared with the control group,ALT and AST in oleanolic acid high dose group were increased,ALP and TBA showed an increasing trend,histopathology presented obvious hepatocyte swelling.In oleanolic acid high dose group,compared with WT mice,ALT,AST,ALP,and TBA in FXR^-/-mice did not change.The result of bile acids in the liver showed that in WT mice,compared with the control group,free bile acids（CA,DCA,UDCA,βMCA）in oleanolic acid high dose group were decreased and conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）were increased;In FXR^-/-mice,compared with the control group,free bile acids（CA,DCA,UDCA,βMCA）in oleanolic acid high dose group was were reduced,and conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）were increased;In oleanolic acid high dose group,compared with WT mice,free bile acids（CA,DCA,UDCA,βMCA）and conjugated bile acids（TCA,TDCA,TUDCA）in FXR^-/-mice were increased,while TCDCA and T-αMCA were decreased.3.After 3 hours of single dose oleanolic acid administration:In WT mice,compared with the control group,ALT in oleanolic acid high dose group was increased,AST and TBA showed an increasing trend,and histopathological showed inflammatory infiltration of hepatocytes.In FXR^-/-mice,compared with the control group,ALT,AST,and TBA did not change in oleanolic acid high dose group,and there was no significant change in liver histopathology.In oleanolic acid high dose group,compared with WT mice,the ALT,AST,and TBA did not change in FXR^-/-mice.The result of bile acids in the liver showed that in WT mice,compared with the control group,the free bile acids（DCA,UDCA,CDCA,α/βMCA）and conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）in oleanolic acid high dose group were increased;In FXR^-/-mice,compared with the control group,the free bile acids（DCA,UDCA,CDCA,α/βMCA）and conjugated bile acids（TCA,TDCA,TUDCA,TCDCA,T-α/βMCA）in oleanolic acid high dose group were increased;In oleanolic acid high dose group,compared with WT mice,the free bile acids（DCA,UDCA,CDCA,αMCA）and conjugated bile acids（TDCA,TUDCA,TCDCA,T-α/βMCA）in FXR^-/-mice were decreased.Conclusion:FXR gene knockout can alleviate oleanolic acid-induced cholestatic liver injury in mice.It may be related to the increase of the mRNA expression of Bsep,Bacs,Baat,Cyp3a11,Cyp2b10,Ephx1,Ugt1a1,Ugt2b5 after FXR knockout,the decrease of conjugated bile acids in the liver and serum,as well as increase of conjugated bile acids in the bile.