Oleanolic Acid and Ursolic Acid Induce UGT1A1 Expression in HepG2 Cells by Activating PXR Rather Than CAR.

Chunhua Xia,Yuqing Xiong,Mingyi Liu,Hong Zhang,Fang He,Fanglan Liu,Na Yao,Caiwen Zeng,Tao Zhan

doi:10.3389/fphar.2019.01111

Chunhua Xia, Yuqing Xiong + Show 7 more

Open Access

https://doi.org/10.3389/fphar.2019.01111

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Abstract

Background: Oleanolic acid (OA) and its isomer ursolic acid (UA) have recently emerged as research foci based on their biologic activities. We previously demonstrated that UA can inhibit the activities of UGT1A3 and UGT1A4, and OA inhibits UGT1A3 activity in liver microsomes. However, whether OA and UA affect the expression of UGT1As in HepG2 cells and the underlying regulatory mechanism remain unclear. Purpose: The present study aimed to explore the effect of OA and UA on the expression of UGT1As in HepG2 cells and the regulatory mechanisms on UGT1A1 based on the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) signaling pathways. Methods: We analyzed the effect of OA and UA on UGT1A expression and on the PXR/CAR regulatory pathway in HepG2 cells, hPXR-silenced HepG2 cells, and hCAR-silenced HepG2 cells by Q-PCR, Western blotting, and dual-luciferase reporter gene assays. Results: In HepG2 cells, OA and UA both significantly induced the expression of UGT1A1, UGT1A3, UGT1A4, and UGT1A9 and upregulated the expression of PXR. However, OA and UA did not affect CAR expression. A dual-luciferase reporter assay showed that OA and UA could markedly promote PXR-mediated UGT1A1 luciferase activity, whereas OA and UA did not affect CAR-mediated UGT1A1 luciferase activity. In hPXR-silenced HepG2 cells, OA and UA did not elevate UGT1A1 activity compared to the control group. However, the expression of UGT1A1 in hCAR-silenced HepG2 cells was markedly elevated compared to the control group or with non-silenced HepG2 cells treated with OA (10, 20, and 40 μM) or UA (10, 20, and 40 μM). Conclusions: OA and UA significantly induce the expression of UGT1A1, UGT1A3, UGT1A4, and UGT1A9 in HepG2 cells, and their induction on UGT1A1 is mediated by PXR activation, not CAR.

Highlights

UDP-glucuronosyltransferases (UGTs) are the major conjugation enzymes of phase II metabolism in vivo, which plays a vital role in maintaining the balance of endogenous metabolites and the removal of exogenous metabolites (Haberkorn et al, 2002; Manevski et al, 2013; Street et al, 2017)
Antibodies used for immunoblot detection, including anti-human UDP-glucuronosyltransferase 1A1 (UGT1A1), UGT1A3, UGT1A4, and UGT1A9 antibody were all from Abcam (Cambridge, MA, USA)
The inductive effect of CITCO (10 μM) on UGT1A1 mRNA and protein expression in hCAR-silenced HepG2 cells was significantly lower than that in normal HepG2 cells (Figures 8A2–C2 and Figures 8A3–C3). These results indicated that Oleanolic acid (OA) and ursolic acid (UA) had no influence on the UGT1A1 target gene mediated by constitutive androstane receptor (CAR)

Summary

Introduction

UDP-glucuronosyltransferases (UGTs) are the major conjugation enzymes of phase II metabolism in vivo, which plays a vital role in maintaining the balance of endogenous metabolites (e.g., bile acids, bilirubin, and steroids) and the removal of exogenous metabolites (e.g., drugs, environmental chemicals, and dietary constituents) (Haberkorn et al, 2002; Manevski et al, 2013; Street et al, 2017). Changes in UGT1A1 activity may lead to alterations in drug metabolism and increased risk for clinical drug–drug interactions. A decrease of UGT1A1 activity reduces glucuronidation of SN-38 (a pharmacologically active metabolite of the anticancer drug, irinotecan), leading to an increased risk for the development of severe irinotecan-associated toxicity (Takano and Sugiyama, 2017). It is important and significant to investigate the regulation of the UGT1A1 gene for the prevention and treatment of bilirubin metabolic disease and clinical drug–drug interactions. Whether OA and UA affect the expression of UGT1As in HepG2 cells and the underlying regulatory mechanism remain unclear. Purpose: The present study aimed to explore the effect of OA and UA on the expression of UGT1As in HepG2 cells and the regulatory mechanisms on UGT1A1 based on the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) signaling pathways

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