Regulation Mechanism Of Oleanolic Acid On The Target Gene Of UDP-glucuronosyltransferase 1A1 Based On PKC/PXR Signaling Pathway

Background:UDP-glucuronosyltransferase 1(UGT1A1)is an important metabolic enzyme for many exogenous drugs and endogenous toxicant bilirubin.The abnormal expression or function of UGT1A1 will lead to the weakening or termination of the body’s glucuronic acid response.Bilirubin cannot be converted into excretable metabolites with high water solubility and high polarity,and then accumulate in the body,causing hyperbilirubinemia.Oleanolic Acid(OA)has been widely used in the treatment of clinical acute icteric hepatitis,viral hepatitis and other diseases,with liver protection,anti-inflammatory and other effects.The previous research of our group has confirmed that oleanolic acid can induce the expression of UGT1A1 in Hep G2 cells by activating PXR.Protein Kinase C(PKC)is an important signaling molecule in the body,activated PKC can inhibit the expression of its downstream target genes by inhibiting the activity of the nuclear receptor PXR.How oleanolic acid regulates the expression of UGT1A1 target genes through the PKC/PXR signaling pathway is still unclear,and it is worthy of further exploration.Objectives:The combination of PKCα and PXR with heat shock protein(HSP90α)and receptor activator protein 1(SRC1)and the expression changes of target gene UGT1A1 intervented by oleanolic acid was studied in normal,over-expressed h PKCαand naphthalene isothiocyanate(ANIT)induced injured Hep G2 cells,which explored the molecular regulation mechanism of oleanolic acid on the target gene UGT1A1 based on the PKC/PXR signaling pathway,and provided a theoretical basis for the treatment of bilirubin metabolism disorders.Methods:1.RT-q PCR,co-immunoprecipitation,immunofluorescence,Western-blot and other experimental techniques were used to explore the effect of OA on the expression of PXR,SRC1,HSP90α and UGT1A1,and investigate the effect of OA on the binding of PKCα or PXR to HSP90α,SRC1 proteins in normal Hep G2 cells.2.In h PKCα over expressed Hep G2 cells,used RT-q PCR,western-blot,and co-immunoprecipitation experimental techniques to study the effect of OA on PKCα,PXR and UGT1A1 expression,as well as the effect on the binding of PKCα or PXR to HSP90α,SRC1 protein through PKC/PXR signaling pathway.3.In ANIT induced injury Hep G2 cells,elisa and western-blot were used to examine the effect of OA on the expression of PKCα,PXR,and target gene UGT1A1 through PKC/PXR signaling pathway.Then explored the possible mechanism of PKC/PXR signaling pathway on target gene UGT1A1 based on OA.Results:1.Effect of OA on the expression of PXR,SRC1,HSP90α and target gene UGT1A1,and its effect on the binding of PKCα or PXR to HSP90α,SRC1 proteins in Hep G2 cells.(1)In normal Hep G2 cells,oleanolic acid obviously up-regulated the expression of target gene UGT1A1 m RNA and protein,the m RNA and protein were increased by76.40% and 47.90% respectively,and also up-regulated the expression of PXR total protein and nuclear protein,but had no significant effect on the total protein expression of SRC1 and HSP90α.(2)The binding of PXR to HSP90α was decreased by 42.87% after oleanolic acid intervention,and the binding of PXR to SRC1 was increased by 43.84%.After PMA(PKC agonist)intervention,the binding of PKCα to HSP90α decreased.Oleanolic acid can antagonize this effect of PMA and then promote the binding of PKCα to HSP90α in Hep G2 cells.2.Effect of OA on the expression of PXR,SRC1,HSP90α and target gene UGT1A1,and its effect on the binding of PKCα or PXR to HSP90α,SRC1 proteins in h PKCα over expressed Hep G2 cells model.(1)In h PKCα over expressed Hep G2 cells model,the expression of PKCαcytoplasmic protein increased,however,there was no difference in the expression of PKCα membrane protein,which would not cause PKCα change of activation state.(2)In h PKCα over expressed Hep G2 cells model,after PMA stimulates PKCαactivation,the binding of HSP90α to PKCα was decreased by 42.00%,binding effect of HSP90α and PXR up-regulated 137.98% in cytoplasm,meanwhile binding effect of PXR and SRC1 was decreased by 41.50% in nucleus,suppressed the protein expression of UGT1A1.When acting with OA,the above effects are antagonized or even reversed.3.Effect of OA on the expression of PKCα,PXR and target gene UGT1A1 in ANIT induced injury Hep G2 cells model.(1)Compared with normal Hep G2 cells,the inflammatory factors such as IL-6and TNF-α were significantly increased,the membrane protein expression of PKCαwas obvious increased,the nuclear protein expression of PXR was obvious decreased,and the target gene UGT1A1 protein expression was decreased by 41.25% in ANIT induced injury Hep G2 cells.(2)In ANIT induced injury Hep G2 cells,Inflammatory factors such as IL-6 and TNF-α were significantly decreased,the expression of PKCα membrane protein was significantly down-regulated,the expression of PXR nuclear protein was significantly up-regulated,and the protein expression of target gene UGT1A1 was significantly up-regulated 64.60% after oleanolic acid intervention.Conclusions:1.In normal and h PKCα-overexpressing Hep G2 cells,PXR and PKCαcompeted to bind the co-inhibitor HSP90α in cytoplasm,Oleanolic acid antagonizes PMA-induced activation of PKCα,inhibited the transfer of PKCα to the cell membrane,and promotes the binding of PKCα to HSP90α in the cytoplasm,thus reduces the binding of PXR to HSP90α,which is conducive to the increase of PXR activation into the nucleus,and then increases the binding of SRC1,ultimately up-regulates the expression of downstream target gene UGT1A1.2.In ANIT induced injury Hep G2 cells,PKCα was activated and transferred to the cell membrane,PKCα in the cytoplasm was decreased,and PXR nuclear protein was also decreased accordingly under inflammatory conditions.Oleanolic acid can play an anti-inflammatory injury effect by inhibiting the activation of PKCα,promoting the entry of PXR into the nucleus and then up-regulating the protein expression of the target gene UGT1A1.