Protective Effects Of Oleanolic Acid On ANIT-induced Intrahepatic Cholestasis By Regulating Bile Acid Transporters And Metabolic Enzymes

Background:Oleanolic acid(OA),a natural pentacyclic triterpenoid compound,was widely distributed in some traditional Chinese herbs such as Hawthorn,Black plum and Fructus Ligustri Lucidi etc.It owns many biological activities including liver,cardiovascular system protection and anti-tumor etc.The effect of OA on liver protection is mainly reflected in the decrease of serum transaminase,thus it clinically applied in treatment of hepatitis.However,There was less report on the effect and mechanism of OA in intrahepatic cholestasis.Bile acid uptake transporter(NTCP),efflux transporter(Bsep,Mrp2 and Mrp3),phase ? metabolizing enzyme(UGT1A1,Sult2a1)and nuclear factor(FXR and Nrf2)are involved in the metabolism of bile acid and have great significance to relieve intrahepatic cholestasis.Objectives:The purpose of this study is to explore the effect of OA in intrahepatic cholestasis and Its mechanism in bile acid metabolism for providing a theoretical basis for the clinical application of OA in intrahepatic cholestasis.Methods:Thirty-five SPF grade male rats(180 g ~220 g)were fed with standard diet and water for 1 week.There were 5 groups(n = 7),as follows:(1)Normal group;(2)model group;(3)OA(50mg/kg)group;(4)OA(100mg/kg)group;(5)UDCA group.All of groups were pretreated for 10 days.On the 10 th day,rats in all groups(except Normal group)was administered with ANIT.On 48 hours after ANIT administration(fasting for 12 hours before and after gavage),blood and liver tissue was collected.1?The blood was collected from the inferior vena cava,serum was separated from blood to evaluate ALT,AST,ALP,TBIL,DBIL,TBA level by biochemical test kits.The HE staining of liver tissue was used to observe the morphology and damage degree of liver tissue.2?The mRNA expression level of sodium taurocholate cotransporter(NTCP),bile salt output pump(Bsep),multidrug resistance protein 2(Mrp2),multidrug resistance protein 3(Mrp3)and phase?metabolizing enzyme(UGT1A1,Sult2a1)in liver were assessed by using Real-time fluorescence quantitative PCR technology.3?The protein expression of sodium taurocholate cotransporter(NTCP),bile salt output pump(Bsep),multidrug resistance protein 2(Mrp2),multidrug resistance protein 3(Mrp3),phase?metabolizing enzyme(UGT1A1,Sult2a1)and nuclear factor(FXR,Nrf2)were detected by Western-blot technology.Results:1.ANIT can significantly increase serum levels of ALT,AST,ALP,TBIL,DBIL and TBA in rats,and increase liver necrosis and inflammation in rats.Both OA and UDCA pretreatment could improve the symptoms of intrahepatic cholestasis by decreasing the level of serum ALT,AST,ALP,TBIL,DBIL,TBA and reducing the necrosis of hepatocytes and inflammatory reaction.Besides,the effect of OA shows dosage association.2.OA pretreatment can reverse the inhibitory effect of ANIT on the mRNA and protein expression of bile acid uptake transporter(NTCP),efflux transporter(Bsep,Mrp2)and phase ? metabolizing enzyme(UGT1A1,Sult2a1)to promote the efflux and metabolic detoxification of bile acid.Bsides,OA pretreatment can also increase the expression of mRNA and protein expression of multi-drug resistance protein(Mrp3)on the basis increasing expression induced by ANIT to alleviate the liver injury for making up for the deficiency of spontaneous adaptive induction of organism.3.OA may be involved in regulating the expression of efflux transporter(Bsep,Mrp2 or Mrp3)through activation of FXR and Nrf2.Conclusion:1.OA has protective effect on ANIT-induced intrahepatic cholestasis.2.OA increases the excretion and metabolism of bile acid by regulating the expression of mRNA and protein of bile acid transporters and phase ? metabolic enzymes to ameliorate ANIT-induced acute intrahepatic cholestasis.3.OA may be involved in the regulation of bile acid efflux transporter(Bsep,Mrp2 or Mrp3)through activation of Nrf2 and FXR to ameliorate ANIT-induced acute intrahepatic cholestasis.