Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes

Abstract

Oleanolic acid is a plant-derived triterpenoid, which protects against various hepatotoxicants in rodents. In order to determine whether oleanolic acid activates nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor known to induce various antioxidant and cytoprotective genes, wild-type and Nrf2-null mice were treated with oleanolic acid (90 mg/kg, i.p.) once daily for 3 days. Oleanolic acid increased nuclear accumulation of Nrf2 in wild-type but not Nrf2-null mice, as determined by Western blot and immunofluorescence. Oleanolic acid-treated wild-type mice had increased hepatic mRNA expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); heme oxygenase-1 (Ho-1); as well as Nrf2 itself. In addition, oleanolic acid increased protein expression and enzyme activity of the prototypical Nrf2 target gene, Nqo1, in wild-type, but not in Nrf2-null mice. Oleanolic acid protected against acetaminophen hepatotoxicity in wild-type mice but to a lesser extent in Nrf2-null mice. Oleanolic acid-mediated Nrf2-independent protection from acetaminophen is, in part, due to induction of Nrf2-independent cytoprotective genes, such as metallothionein. Collectively, the present study demonstrates that oleanolic acid facilitates Nrf2 nuclear accumulation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.

Fig 1

Upper. Western blot of liver nuclear fractions for Nrf2 after treatment with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Also shown is the quantification of specific band intensity, normalized to β-actin, and expressed relative to control as mean ± S.E.M. Abbreviations: ND, Not Detected; Veh, Vehicle; OA, oleanolic acid; WT, wild-type; null, Nrf2-null. Asterisks (*) indicate a statistically significant difference from wild-type mice treated with vehicle (p ≤ 0.05). Lower. Immunofluorescent localization of Nrf2 in livers from wild-type mice after vehicle or oleanolic acid. Indirect immunofluorescence to detect Nrf2 (green) and actin (red) was performed on liver cryosections (5 μm) from wild-type mice after oleanolic acid (90 mg/kg, i.p.) treatment once daily for three days. Sections were mounted in Prolong Gold containing DAPI for nuclear staining (blue). Representative images are shown at high-power magnification (×400). Bar represents 200 μm.

Fig 2

Messenger RNA expression of Nqo1, Gclc, and Ho-1 in livers of wild-type and Nrf2-null mice after treatment with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Messenger RNA was quantified by the bDNA assay. Data is expressed relative to wild-type controls as mean ± S.E.M. Abbreviations: WT, wild-type; null, Nrf2-null. Asterisks (*) indicate a statistically significant difference from wild-type mice receiving vehicle (p ≤ 0.05).

Fig 3

Upper. Protein expression of Nqo1 determined by western blot in livers of wild-type and Nrf2-null mice after treatment with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Also shown is the quantification of specific band intensity, normalized to β-actin, and expressed relative to control as mean ± S.E.M. Lower. Nqo1 enzyme activity in livers of wild-type and Nrf2-null mice after treatment with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Units are in nmole DCPIP reduced per min per mg of protein expressed as mean ± S.E.M. Abbreviations: Veh, Vehicle; OA, oleanolic acid; WT, wild-type; null, Nrf2-null. Asterisks (*) indicate a statistically significant difference from wild-type mice receiving vehicle (p ≤ 0.05).

Fig 4

Serum alanine transaminase (ALT) levels in wild-type and Nrf2-null mice pretreated with oleanolic acid (90 mg/kg, i.p.) once daily for three days (n=5 for all groups except Nrf2-null mice receiving vehicle, n=4), administered acetaminophen (500 mg/kg, i.p.) on the fourth day, and sacrificed 8-h later. Units are in International Units/Liter (IU/L) expressed as mean ± S.E.M. A minus sign (-) represents that the animal was administered the appropriate vehicle for the compound specified. A plus sign (+) represents that the animal was administered the compound specified. Asterisks (*) indicate a statistically significant difference from wild-type mice receiving vehicle (p ≤ 0.05). Daggers (†) indicate a statistically significant difference from Nrf2-null mice receiving vehicle (p ≤ 0.05).

Fig 5

Messenger RNA expression of MT from livers of wild-type and Nrf2-null mice after treatment with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Messenger RNA was quantified by the bDNA assay. Data is expressed relative to wild-type controls as mean ± S.E.M. Asterisks (*) indicate a statistically significant difference from wild-type mice receiving vehicle (p ≤ 0.05).