Induction Mechanism Of Oleanolic Acid And Ursolic Acid On UDP-glucuronosyltransferase 1A1 Based On Pregnane X Receptor And Constitutive Androstane Receptor Regulatory Pathway

Background:Oleanolic acid and ursolic acid are pentacyclic triterpenoid isomers,which possess several pharmacological effects,such as anti-inflammatory,hepatoprotection,hypoglycemic and anti-tumor effects.OA is widely used for treating acute and chronic liver injury in clinical.Previous studies have shown that UA can inhibit the activities of UGT1A3 and UGT1A4,and OA inhibits UGT1A3 activity in liver microsomes.However,it is not clear whether OA and UA affect the expression of UGT1 As target gene in human hepatocytes and whether they are regulated by the pregnane X receptor(NR1I2;PXR)and constitutive androstane receptor(NR1I3;CAR),which are worthy of further study and discussion.Objectives:The present study aimed to explore the effect of OA and UA on the expression of UGT1 As in HepG2 cells and the induction mechanisms on UGT1A1 based on the pregnane X receptor and constitutive androstane receptor regulatory pathways,which provided experimental basis for guiding rational clinical medication of OA and UA.Methods:1.The effect of OA and UA on PXR,CAR and UGT1 As mRNA and protein expression were investigated by RT-qPCR,Western blot and other experimental techniques.2.The effect of OA and UA on the activity of UGT1A1 reporter gene in HepG2 cells transiently transfected with hPXR or hCAR was investigated by dual-luciferase reporter assay.3.ShRNA interference technique was used to examine the effect of OA and UA on UGT1A1 mRNA and protein expression in the hPXR/hCAR-silenced HepG2 cells.Result:1.Effect of OA and UA on the expression of UGT1As?PXR and CAR in HepG2 cells(1)OA(10,20,and 40 ?M)significantly upregulated UGT1A1,UGT1A4 and PXR mRNA and protein levels in a concentration-dependent manner.Meanwhile,OA significantly induced UGT1A3 and UGT1A9 mRNA and protein expression.Specifically,20 ?M OA exhibited a highest inductive effect on UGT1A3 and UGT1A9.(2)UA(10,20,and 40 ?M)significantly upregulated UGT1A1,UGT1A3,UGT1A4,UGT1A9 and PXR mRNA and protein levels in a concentration-dependent manner.(3)Both OA and UA had no detectable effect on expression of CAR mRNA and protein.2.Effect of OA and UA on the UGT1A1 reporter gene in HepG2 cells transiently transfected with hPXR or hCAR(1)In HepG2 cells transiently transfected with hPXR,both OA and UA could significantly upregulate the PXR-mediated UGT1A1 transcriptional activity after 24 h intervention in a concentration-dependent manner.(2)In HepG2 cells transiently transfected with hCAR,OA and UA had almost no significant impact on the CAR-mediated UGT1A1 transcriptional activity after 24 h intervention.3.Effect of OA and UA on the expression of UGT1A1 in hPXR/hCAR-silenced HepG2 cells(1)In the hPXR-silenced HepG2 cell model,OA and UA exhibited a weaker induction on UGT1A1 mRNA and protein expression compared with in HepG2 cells.(2)In the hCAR-silenced HepG2 cell model,OA and UA had no obvious effect on the induction of UGT1A1 mRNA and protein compared with in HepG2 cells.Conclusions:1.In HepG2 cells,both OA and UA could significantly upregulate the expression of UGT1A1,UGT1A3,UGT1A4,UGT1A9 and PXR mRNA and protein.However,OA and UA had no significant effect on the expression of CAR mRNA and protein.2.The induction effect of OA and UA on UGT1A1 in HepG2 cells was mainly related to the PXR rather than CAR regulatory pathway.