High Fat Diet-Induced Hepatic 18-Carbon Fatty Acids Accumulation Up-Regulates CYP2A5/CYP2A6 via NF-E2-Related Factor 2.

Mouse hepatic pathology. Liver sections of the former four figures were stained by hematoxylin and eosin. Liver sections of the latter four figures were stained with Oil Red O and the fat accumulated in hepatocytes manifested red. All the liver sections were examined by light microscopy and the images were displayed at 200× the original magnification. The fat accumulation in hepatocytes were pointed out by the arrows in HFD groups.

Nrf2 and CYP2A5 protein expressions in HFD feeding mice. Nrf2 protein expression in mouse hepatocytes nucleus and its quantity relative to Histon H1 is shown in panel (A). Nrf2 protein expression in liver cytoplasm and its quantity relative to β-actin is shown in panel (B). Hepatic CYP2A5 protein expression in WT and Nrf2-null mice, which fed with CD and HFD, is revealed in panel (C) and the quantity relative to β-actin is shown in panel (D). * represent statistical difference caused by HFD within WT or Nrf2-null groups; $ represent statistical difference caused by Nrf2 deficiency on the same diet. * 0.01 < P < 0.05, **P < 0.01; $ 0.01 < P < 0.05, $$P < 0.01.

Effect of 18-carbon fatty acid on Nrf2 and CYP2A6 expression in HepG2 cells. (A) HepG2 cells viability which were administered with increasing concentrations of SA, OA, LA, and ALA. The cells viability was represented by the absorbance. The effect of SA, OA, LA, and ALA on Nrf2 and CYP2A6 protein expression in HepG2 cells are exhibited in panel (B). Nrf2 protein expression was quantified and shown in panel (C). CYP2A6 protein expression was quantified and shown in panel (D). The effect of SA, OA, LA, and ALA on Nrf2 and CYP2A6 mRNA expressions are shown in panel (E,F), respectively. Asterisks * represent statistical difference caused by fatty acid administration from control cells. * 0.01< P < 0.05, **P < 0.01.

Nrf2 gene silence and over expression. In Nrf2 siRNA transfected HepG2 cells, Nrf2 protein expression is shown in panel (A) and its quantity relative to β-actin is shown in panel (B); Nrf2 mRNA expression is shown in panel (C). In pcDNA3-EGFP-C4-Nrf2 transfected HepG2 cells, Nrf2 protein expression is shown in panel (D) and its quantity relative to β-actin is shown in panel (E); Nrf2 mRNA expression is shown in panel (F). The GSTA1 mRNA relative expression in different groups was shown in panel (G). NC, in panel (A–C) represents the HepG2 cells that transfected with negative control siRNA, in panel (D–F) represents the HepG2 cells that transfected with negative control plasmid (pcDNA3-EGFP-C4). In panel (G), NC-O represents the cells that transfected with pcDNA3-EGFP-C4 and NC-S represents the HepG2 cells that transfected with negative control siRNA. Cell, means the control cells that without any administration. Asterisks * represent statistical difference caused by Nrf2 siRNA or pcDNA3-EGFP-C4-Nrf2 transfection from control cells without any stimulation. * 0.01< P < 0.05, **P < 0.01.

CYP2A6 and GSTA1 expression changes in Nrf2 silenced and over-expressed HepG2 cells administered with LA and ALA. CYP2A6 mRNA (A) and protein (B,C) expressions were both decreased significantly in Nrf2 silenced cells with or without LA and ALA treatment. CYP2A6 mRNA (D) and protein (E,F) expressions were increased in Nrf2 over-expressed cells with or without LA and ALA administration. In the presence or absence of LA and ALA administration, GSTA1 mRNA expression was down-regulated in cells transfected with Nrf2 siRNA (G) and was up-regulated in cells transfected with pcDNA3-EGFP-C4-Nrf2 (H). In addition, the NC cells in panel (A–C,G) were transfected with NC siRNA. The NC cells in panel (D–F,H) were transfected with NC plasmid (pcDNA3-EGFP-C4). * represent statistical difference caused by transfection of Nrf2 siRNA and pcDNA3-EGFP-C4-Nrf2 from cells transfected with NC siRNA and NC plasmid; $ represent statistical difference caused by LA and ALA administration within Nrf2 silenced cells, Nrf2 over expressed cells, and control cells. *0.01 < P < 0.05, **P < 0.01; $ 0.01 < P < 0.05, $$P < 0.01.