Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis

Abstract

Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqol mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.

Fig 1.

Total hepatic RNA was isolated from mice that underwent sham or BDL surgery at days 1, 3, and 7 and analyzed by the bDNA assay for Nqo1, Ho-1, Gst a1, a4, m1, m2, m3 mRNA expression. The data are presented as mean relative light units ± standard error of the mean (n = 3–6 animals). Asterisks (*) represent a statistical difference (P < 0.05) between sham and BDL groups

Fig 2.

Nqo1 protein expression and activity in liver cytosolic fractions from mice 1, 3, and 7 days after sham or BDL surgery. (A) Representative Western blot of liver cytosolic fractions from mice that underwent sham surgery or BDL surgery (days 1, 3, and 7) stained with anti-Nqo1 antibodies. (B) Quantification of Nqo1 protein levels in liver cytosolic fractions from mice that underwent sham or BDL surgery. The data are presented as relative protein expression ± standard error of the mean (SEM) (n = 5–13 animals). (C) The data are presented as nanomoles of reduced DCPIP per minute per milligram of protein ± SEM (n = 5–6 animals). Asterisks (*) represent a statistical difference (P < 0.05) between sham and BDL groups

Fig 3.

Expression and activity of Nqo1 in liver cytosolic fractions from wild-type and Nrf2-null mice 1 and 3 days after sham or BDL surgery. (A) Total RNA was isolated from livers of WT and Nrf2-null mice that underwent sham or BDL surgery 1 or 3 days previously, and analyzed by the bDNA assay for Nqo1 mRNA expression. Data from sham animals at 1 and 3 days were not statistically different and were pooled and denoted as sham. The data are presented as mean relative light units ± standard error of the mean (SEM) (n = 4–7 animals). (B) Upper panel: Representative Western blot of liver cytosolic fractions from mice 3 days after sham or BDL surgery (n = 4–7 animals) stained with anti-Nqo1 antibodies. Lower panel: Quantification of Nqo1 protein levels in liver cytosolic fractions from mice 1 and 3 days after sham or BDL surgery. The data are presented as relative protein expression ± SEM (n = 4–7 animals). (C) Analysis of Nqo1 activity in liver cytosolic fractions from WT and Nrf2-null mice that underwent sham or BDL surgery 1 or 3 days previously. The data are presented as nanomoles of DCPIP reduced per minute per milligram of protein ± SEM (n = 4–7 animals). Asterisks (*) represent a statistical difference (P < 0.05) between WT sham and WT BDL groups, and crosses (†) represent a statistical difference between WT and Nrf2-null mice undergoing the same type of surgery

Fig 4.

Histologic examination of liver sections and analysis of hepatic bile acid accumulation from wild-type and Nrf2-null mice 3 days after BDL. Portions of liver from wild-type and Nrf2-null mice 3 days after sham or BDL surgery were fixed in formalin and stained with hematoxylin and eosin. Sham-operated wild-type (A) and Nrf2-null (B) mouse liver demonstrated normal tissue histology (4× magnification). BDL resulted in multifocal hepatocellular necrosis (arrows) in wild-type (C, E) and Nrf2-null (D, F) mice. Hepatocyte degeneration and necrosis is seen at 4× (C, D) and 10× (E, F) magnification. (G) Determination of liver bile acid concentration (nanomoles per gram of tissue). Values are expressed as mean ± standard error of the mean. Asterisks (*) represent a statistical difference (P < 0.05) between sham and BDL groups for a respective genotype. Crosses (†) represent a statistical difference (P < 0.05) between wild-type and Nrf2-null mice undergoing BDL