Effects And Mechanisms Of Oleanolic Acid On Alleviating Cholestatic Liver Injury Via NRF2/FXR Dual Signaling Pathway

Background:Cholestasis is a clinical syndrome in which the metabolism and circulation of bile acids are impaired.Currently,treatments have limited clinical efficacy.Therefore,it is particularly important to further study the regulation mechanism of bile acid metabolism and transport and find new therapeutic strategies.Farnesol X receptor（FXR）and nuclear factor red related factor 2（NRF2）are two important factors that regulate the expression of bile acid metabolic enzymes and transporters,and are important targets for regulating bile acid homeostasis and treating cholatic liver injury.The existing anti-cholestatic pharmacological studies often involve only a single regulatory factor or a single target,and pay little attention to the interaction and co-regulation of FXR and NRF2 signaling pathways,which is obviously not conducive to the clinical treatment of anti-cholestatic liver injury.Oleanolic acid（OA）has biological activities such as liver protection.Some scholars used bile duct ligation and cholestatic acid to build models,starting from NRF2 and FXR single signal pathways respectively,and studies showed that OA had a good anti-cholestasis effect.Then,does OA jointly regulate the balance of bile acid metabolism and prevent cholestatic liver injury through NRF2 and FXR dual signaling pathways?It still needs to be clarified and further explored.The biliary tract characteristics ofα-naphthol isothiocyanate（ANIT）-induced cholestasis in rodents are more similar to the pathological characteristics of human primary bile duct cholangitis,so it is necessary to use ANIT-induced cholestatic liver injury model to further study the anti-cholestatic effect of OA.Other studies have suggested thatβ-catenin is involved in the process of cholestasis under inflammation,and inhibition ofβ-catenin can activate FXR,thereby reducing bile acid deposition in the liver.β-catenin knockdown promotes NRF2 activation and oxidative stress response.Therefore,we speculated that during the activation of NRF2 and FXR dual signaling pathways by OA,the two signaling pathways may interact throughβ-catenin/P300 to cooperatively regulate the expression of downstream target genes.Objectives:In this study,a model of ANIT-induced cholestatic liver injury was established,and technological means such as bile acid targeted metabolomics,gene knockout or silencing,and immunoprecipitation were adopted to focus on the co-regulation and interaction of NRF2 and FXR dual signaling pathways,and to study the protective effect of OA on ANIT-induced cholestatic liver injury and its mechanism.It provides theoretical basis and scientific basis for multi-target combination therapy and multi-target drug development in clinical cholestasis.Methods:1.Protective effect of OA on cholestatic liver injury in rats:Sprague Dawley（SD）rats were divided into normal control group,OA administration group alone,ANIT model group,ursodeoxycholic acid（UDCA）positive control group,low,medium and high dose OA intervention groups,and UDCA+OA combined intervention group.The rats were given continuous gavage for 7 d,and ANIT model was given gavage 48 hours before death.Biochemical indexes such as ALT,AST and GSH were determined,histopathological changes of liver were observed by HE staining,and bile acid changes were analyzed by targeted metabonomics.The expressions of Nrf2,Fxr and their downstream Bsep,Ntcp,Cyp7a1 and Ugt1a1 genes in rat liver tissues were determined by RT-q PCR and western blot.Immunohistochemistry was used to investigate the protein expression of BSEP,NTCP,CYP7A1 and UGT1A1 in liver tissue.2.Protective effect of OA on rat primary hepatocytes and its effect on NRF2 and FXR dual signaling pathway:Rat primary hepatocytes were isolated by two-step in situ perfusion method.Nrf2 or Fxr genes were silenced by RNA interference technique.ANIT modeling and OA intervention were used to determine the activity of rat primary hepatocytes and biochemical indexes such as ALT,AST and LDH.The expressions of Nrf2,Fxr,Bsep,Ntcp,Cyp7a1 and Ugt1a1 genes in hepatocytes were determined by RT-q PCR and western blot.3.Protective effect of OA on cholestatic liver injury in Nrf2 gene knockout（Nrf2-/-）rats and Fxr gene silenced（Fxr-kd）rats:Rats were divided into normal control group,ANIT model group and OA intervention group.The rats were intragastric administrated for 7 d continuously.The rats were gavaged 48 h before death by ANIT for modeling.ALT,AST,GSH and other biochemical indexes were determined,and histopathological changes of liver were observed by HE staining.The changes of bile acids were analyzed by targeted metabolomics.The gene expressions of Nrf2,Fxr and their downstream targets Bsep,Ntcp,Cyp7a1 and Ugt1a1 in rat liver tissues were determined by RT-q PCR and western blot.Immunohistochemistry was used to investigate the protein expression of BSEP,NTCP,CYP7A1 and UGT1A1 in liver tissue.4.Interaction of NRF2 and FXR dual signaling pathways in L02 cells:NRF2 or FXR genes of L02 cells were silenced by RNA interference technique.ANIT modeling and OA intervention were used to determine the activity of L02 cells and biochemical indexes such as ALT,AST and LDH.The expressions of NRF2,FXR,BSEP,NTCP,CYP7A1 and UGT1A1 genes in L02 cells were determined by RT-q PCR and western blot.5.Interaction between NRF2 and FXR in L02 cells:Discovery Studio was used for mapping and molecular docking technology was used to evaluate the interaction between OA and Kelch-like ECH-associated protein l（KEAP1）and FXR proteins.RNA interference technique was used to silence NRF2 or FXR genes in cells,and Co-immunoprecipitation was used to study the effect of OA on the binding of NRF2 to KEAP1 and FXR to RXRα.The effects of SFN and GW4064 on the binding of NRF2and P300,P300 andβ-catenin,β-catenin and FXR,FXR and RXRα.Results:1.Protective effect of OA on cholestatic liver injury in rats:（1）OA（25-100 mg/kg）could dose-dependently reverse the elevation of ALT,AST,ALP,TBIL,γ-GT and TBA in serum induced by ANIT,and increase GSH and SOD in liver tissue,and relieve the bleeding,necrosis and inflammatory infiltration of ANIT induced rat liver.In the high-dose OA group,bleeding,necrosis and inflammatory infiltration in the portal area almost disappeared,and the morphology of liver tissue was basically normal.（2）High-dose OA group could promote bile acid metabolism and transport,decrease bile acid content in serum and liver,and increase faeces bile acid content in rats with cholestatic liver injury.Before and after intervention,PCA evaluation of bile acids in serum,liver and faeces of rats were completely separated.（3）OA（25-100 mg/kg）can increase the mRNA and protein expressions of Bsep,Ntcp and Ugt1a1,and decrease the gene level of Cyp7a1 in hepatic tissue of rats with cholestatic liver injury.Significantly increased the expression level of Fxr mRNA and protein,promoted NRF2 into the nucleus and Nrf2 mRNA expression.（4）Compared with the UDCA group,the combined treatment of OA（100 mg/kg）could significantly reduce the serum ALT and AST,and increase the accumulation of GSH and NRF2 nuclei in the liver of the rats with cholatic liver injury.2.Protective effect of OA on rat primary hepatocytes and its effect on NRF2 and FXR dual signaling pathway:（1）Compared with normal control group,OA can up-regulate Nrf2 and Fxr gene levels,increase Bsep and Ugt1a1mRNA and protein levels,decrease Cyp7a1 gene expression,and almost have no effect on Ntcp gene expression.（2）Compared with ANIT model group,OA significantly increased the activity,GSH and SOD of liver cells,and decreased ALT,AST and LDH of cell supernatant.（3）Compared with ANIT model group,OA decreased Cyp7a1 gene expression,reversed the decrease of Bsep,Ntcp,Ugt1a1 mRNA and protein levels,significantly upregated Fxr gene level,and increased NRF2 protein expression in cell nucleus.（4）Nrf2 or Fxr gene silencing significantly weakened OA’s protective effect on ANIT-treated hepatocytes and its regulation on downstream target genes.3.Protective effect of OA on cholestatic liver injury in Nrf2-/-rats and Fxr-kd rats:（1）Compared with normal control group of Nrf2-/-rats and Fxr-kd rats,serum ALT,AST,ALP,TBIL,γ-GT and TBA in ANIT model group were significantly increased,liver GSH and SOD were significantly decreased,and large area of bleeding,necrosis and inflammatory infiltration occurred in liver tissue.（2）Compared with ANIT model group,the levels of ALT,AST and ALP in serum of Nrf2-/-rats in OA（100 mg/kg）intervention group were significantly decreased,while the levels of TBIL,γ-GT and TBA were not significantly changed.GSH and SOD in liver were significantly increased.Bleeding and necrosis areas in liver tissue were significantly decreased.But there was still a lot of inflammatory infiltration in the portal area.Compared with ANIT model group,the levels of ALT,AST,ALP,TBIL,γ-GT and TBA in serum of fxr-kd rats in OA intervention group were significantly decreased,while the levels of GSH and SOD in liver were significantly increased.There was no obvious bleeding and necrosis area in liver tissue,and the inflammatory infiltration area in portal area decreased.（3）Compared with ANIT model group,OA could not significantly affect the contents of bile acids in serum,liver and faeces of Nrf2-/-rats.But could significantly increase the mRNA and protein levels of Fxr and Bsep in liver,and significantly decrease the mRNA and protein levels of Cyp7a1.However,NTCP and UGT1A1protein expressions were not significantly affected.Compared with ANIT model group,OA could promote bile acid metabolism and transport,decrease bile acid content in serum and liver,and increase faeces bile acid content in fxr-kd rats.But some bile acids abundance did not change significantly.It can significantly increase the mRNA levels of Nrf2,Fxr,Besp and Ugt1a1 in liver,significantly reduce the mRNA and protein levels of Cyp7a1,and promote the nuclear accumulation of NRF2 and protein expressions of FXR,BSEP and UGT1A1.（4）Compared with the experimental results of WT rats,OA significantly reduced the improvement of biochemical parameters and liver pathological tissue of anit-induced Nrf2-/-rats,and had no significant effect on the change of bile acid metabonomics induced by anit-induced Nrf2-/-rats,and significantly reduced the regulation of bile acid metabolic transporter expression.Compared with the experimental results of WT rats,OA had a weaker improvement effect on hepatic pathological tissue of anit-induced Fxr-kd rats,failed to completely reverse the metabolic changes of bile acid induced by ANIT,and had a relatively weaker effect on the expression of bile acid metabolic transporter.4.The protective effect of OA on L02 cells and its effect on NRF2 and FXR dual signaling pathways:（1）Compared with normal control group,OA up-regulated NRF2and FXR gene levels in L02 cells,increased BSEP and UGT1A1 mRNA and protein levels,and decreased CYP7A1 gene expression.（2）Compared with ANIT model group,OA significantly increased the activity of L02 cells,and decreased ALT,AST,LDH in cell supernatant.（3）Compared with ANIT model group,OA decreased CYP7A1 gene expression in L02 cells,reversed the decrease of BSEP,NTCP,UGT1A1 mRNA and protein levels,significantly upregated FXR gene level,and increased NRF2 protein expression in cell nucleus.（4）NRF2 or FXR gene silencing significantly reduced the protective effect of OA on ANIT-induced L02 cell injury and its regulation on downstream target genes,in which NRF2 gene silencing had a more significant effect on OA.5.Interaction of NRF2 and FXR dual signaling pathways in L02 cells:（1）The docking score of OA and KEAP1 protein molecule was-8.3 kcal·mol^-1.OA and KEAP1 had strong spatial binding force and could form stable complex.The docking score of OA and FXR was-6.9 kcal·mol^-1,indicating that OA had a binding effect with FXR.（2）OA significantly reduced the binding of NRF2 to KEAP1 and increased the binding of FXR to RXRαin L02 cells.（3）In ANIT-induced L02 hepatocyte injury model,the binding of NRF2 to P300 decreased,the binding ofβ-catenin to P300 andβ-catenin to FXR increased significantly,and the binding of FXR to RXRαdecreased.（4）SFN or GW4064 can promote the binding of NRF2 to P300,and reduce the binding ofβ-catenin to P300 andβ-catenin to FXR.Conclusion:1.In the model of ANIT-induced cholestatic liver injury in rats,OA can significantly reduce the synthesis of bile acid,promote the metabolism and flow of bile acid,improve the homeostasis of bile acid in injured rats,and significantly alleviate the cholestatic liver injury caused by ANIT.After Nrf2 gene was knocked out,OA significantly weakened the regulation and protection of bile acid in ANIT-induced model rats.After Fxr gene silencing,the regulation and protection of OA on bile acid in ANIT-induced model rats were weakened,but OA still significantly alleviated cholestatic liver injury in rats.These results suggest that the ANIT-cholestasis effect of OA is related to the activation of NRF2 and FXR signals and the regulation of the expression of bile acid metabolism and transport related proteins.And NRF2 signaling pathway plays a more important role.2.In ANIT-induced primary hepatocytes and L02 cells,OA can significantly improve hepatocyte injury and regulate the expression of genes related to bile acid metabolism and transport by activating NRF2/FXR dual signaling pathway.However,Silencing NRF2 or FXR gene weakened the regulation of OA on genes related to bile acid metabolism and transport.These results suggest that OA can still play a role through the interaction of NRF2/FXR dual signaling pathway.And NRF2 signaling pathway plays a more important role.3.KEAP1,FXR and OA can interact with each other.There is aβ-catenin/P300mediated interaction mechanism between NRF2 and FXR dual signaling pathways during their activation.