| Background:Ursolic acid and Oleanolic acid are mutually isomeric with pharmacological effects of anti-inflammatory and hepatoprotective.In the process of acute liver injury,liver cells are damaged,and the biliary is excreted disorderly.Bile acids in the human body are mainly metabolized by the glucuronidation of Ⅱ-phase metabolizing enzyme UGT2B7 and excreted by efflux transporters including bile salts output pump(BSEP)and multidrug resistance-associated proteins(MRPs).UA and OA could activate the nuclear receptor Nrf2 to regulate the expression of its downstream genes like antioxidant defense factors related genes,ultimately achieve anti-oxidation,liver protection and other effects.So is the hepatoprotective effect of UA and OA achieved through regulating the expression of UGT2B7,BSEP and MRP2 mediated by Nrf2? It is worth exploring.Objectives:To study the effects of UA and OA on the expression of Nrf2、UGT2B7、BSEP and MRP2 and its mechanism in the HepG2 cells,these will provide more theoretical basis for the hepatoprotective mechanism of UA and OA.Methods:1.The effects of different concentrations of UA and OA on the expression levels of mRNA and protein of Nrf2,UGT2B7,BSEP,and MRP2 at different times were examined by Rt-qPCR and Western bloting in HepG2 cells.2.The effects of UA and OA on the expression levels of mRNA and protein of Nrf2,UGT2B7,BSEP,and MRP2 were studied in Nrf2 silenced HepG2 cell model constructed by transient co-transfection.3.Constructing plasmid,transient co-transfection and dual-luciferase reporter gene assays to explore the mechanisms of UA and OA on Nrf2-UGT2B7/BSEP /MRP2 regulatory pathways.Results:1.In the HepG2 cells,the regulated of UGT2B7 and BSEP by UA/OA had a time-dependent relationship,and the expression of Nrf2,UGT2B7,BSEP and MRP2 at mRNA and protein level were up-regulated most significantly treated with UA and OA at a concentration of 32 μM for 48h:(1)There were significantly up-regulated of the mRNA and protein expression of Nrf2 but no significant effect on the mRNA and protein expression of BSEP and UGT2B7 after treatment of UA for 24 h.(2)After treatment of OA for 24 h,the mRNA expressions of Nrf2,UGT2B7,BSEP and MRP2 were significantly up-regulated,as well as the protein expressions of Nrf2 and MRP2 were significantly up-regulated,but had no effect on UGT2B7 and BSEP protein expression.(3)When the intervention time of UA was prolonged to 48 hours,the mRNA and protein expression of Nrf2,UGT2B7,BSEP and MRP2 were significantly up-regulated.(4)When the intervention time of OA was prolonged to 48 hours,the mRNA and protein expression of Nrf2,UGT2B7,BSEP and MRP2 were significantly up-regulated.2.In the HepG2 cell model that silenced Nrf2,the up-regulation of UGT2B7 and BSEP mRNA and protein was similar to that of normal HepG2 cells,but the up-regulation of MRP2 mRNA and protein was significantly weaked.3.Both UA and OA could significantly induce MRP2 reporter enzyme activity.Conclusions:1.The mRNA and protein expression of Nrf2,UGT2B7,BSEP and MRP2 were significantly up-regulated in HepG2 cells treated with UA and OA,and the up-regulation of Nrf2 was the most significant.2.The up-regulation of MRP2 mRNA and protein was significantly weaked in the HepG2 cell model that silenced Nrf2,but the up-regulation of UGT2B7 and BSEP mRNA and protein was not changed.It suggests that the expression of MRP2 is regulated by Nrf2.The role of UA and OA in promoting the excretion of bile acids may through up-regulate the expression of MRP2 by activating Nrf2.3.Dual luciferase reporter assays confirmed that UA and OA could up-regulate the expression of MRP2 through activating Nrf2. |
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