Low Dose of Oleanolic Acid Protects against Lithocholic Acid–Induced Cholestasis in Mice: Potential Involvement of Nuclear Factor-E2-Related Factor 2-Mediated Upregulation of Multidrug Resistance-Associated Proteins

Abstract

Oleanolic acid (OA) is a natural triterpenoid and has been demonstrated to protect against varieties of hepatotoxicants. Recently, however, OA at high doses was reported to produce apparent cholestasis in mice. In this study, we characterized the protective effect of OA at low doses against lithocholic acid (LCA)-induced cholestasis in mice and explored further mechanisms. OA cotreatment (5, 10, and 20 mg/kg, i.p.) significantly improved mouse survival rate, attenuated liver necrosis, and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; more importantly, serum total bile acids and bilirubin, as well as hepatic total bile acids were also remarkably reduced. Gene and protein expression analysis showed that hepatic expression of multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4 was significantly increased by OA cotreatment, whereas other bile acid metabolism-and transport-related genes, including Na+/taurocholate cotransporter, organic anion transporter 1b2, bile salt export pump, multidrug resistance protein 3, Cyp3a11, Cyp2b10, Sulfotransferase 2a1 (Sult2a1), and UDP-glucuronosyltransferase 1a1 (Ugt1a1), were only slightly changed. OA also caused increased nuclear factor-E2-related factor (Nrf2) mRNA expression and nuclear protein accumulation, whereas nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor were not significantly influenced by OA. Luciferase (Luc) assays performed in HepG2 cells illustrated that OA was a strong Nrf2 agonist with moderate PXR and weak FXR agonism. Finally, in mouse primary cultured hepatocytes, OA dose-and time-dependently induced expression of Mrp2, Mrp3, and Mrp4; however, this upregulation was abrogated when Nrf2 was silenced. In conclusion, OA produces a protective effect against LCA-induced hepatotoxicity and cholestasis, possibly due to Nrf2-mediated upregulation of Mrp2, Mrp3, and Mrp4.

Oleanolic acid attenuates obstructive cholestasis in bile duct-ligated mice, possibly via activation of NRF2-MRPs and FXR antagonism

“…Previously we reported OA exerted hepatoprotection against lithocholic acid (LCA)-induced cholestasis in mice, and NRF2-mediated upregulation of three multidrug resistance-associated proteins (MRP2, MRP3 and MRP4) may represent the main mechanism of its anticholestatic effect (Chen et al, 2014). It is noteworthy that, OA-induced MRP3 and MRP4 expression is more robust than that found in MRP2, meanwhile, MRP3 and MRP4 are the predominant bile acid export pumps located at basolateral membrane of hepatocytes, responsible for subsequent bile acid output in the form of urine.…”

Dynamic and Coordinated Regulation of KEAP1-NRF2-ARE and p53/p21 Signaling Pathways Is Associated with Acetaminophen Injury Responsive Liver Regeneration

“…Western blot analysis was performed as described in our previous report (Chen et al, 2014). Briefly, total liver or nuclear protein extracts were prepared from frozen liver tissues, and protein concentration was determined using the bicinchoninic acid method (Thermo Scientific, Rockford, IL).…”

Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway

“…Malondialdehyde (MDA) levels in the liver were assessed using a commercially available assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Jiang YM et al Acta Pharmacologica Sinica npg Quantitative real-time PCR analysis of Nrf2 Total RNA was isolated from the mouse liver samples using TRIzol reagent according to the manufacturer's instruction (Invitrogen, Grand Island, NY, USA) and quantified using a NanoDrop spectrophotometer (Thermo Scientific, Rockford, IL, USA) as described in a previous report [15] . cDNA synthesis was performed with 1 μg of total RNA using a PrimeScript RT reagent kit with gDNA eraser (TaKaRa Biotech, Kyoto, Japan).…”

“…Western blot analyses were performed as described in our previous reports [11,15] . Briefly, protein extracts were prepared from the liver tissue using RIPA lysis buffer (Biocolors, Shanghai, China) or a Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions.…”