Table 1.
Composition of the experimental diets.
Table 2.
Fatty acid composition (wt%) of dietary fats.
Figure 1.
Effects of fish-oil diet on HO-1 expression and endothelium-dependent vasorelaxation in thoracic aortas.
Control or fish-oil diet was fed to C57BL/6 or Nrf2−/− mice for 3 weeks. (A) The relative mRNA expressions of HO-1 in thoracic aortas were analyzed quantitatively using real-time RT-PCR. Each value represents the mean ± SE of 6–10 animals. (B) Total cell lysates from thoracic aortas were subjected to western blotting analyses. Each value represents the mean ± SE of four animals. (C–F) Concentration-vasodilatory response curves induced by ACh (C, D) or SNP (E, F) in aortic rings obtained from C57BL/6 (C, E) or Nrf2−/− mice (D, F) fed with fish-oil diet for 3 weeks. Each value represents the mean ± SE of 12–18 rings. *P<0.05, **P<0.01, compared with control diet-fed C57BL/6 mice. ††P<0.01, compared with fish-oil diet-fed C57BL/6 mice.
Table 3.
Body weight changes of C57BL/6 (n = 10) and Nrf2−/− mice (n = 6) fed with fish-oil diet for 3 weeks.
Table 4.
Plasma characteristics of C57BL/6 (n = 10) and Nrf2−/− mice (n = 6) fed with fish-oil diet for 3 weeks.
Figure 2.
Effects of fish-oil diet on concentrations of 4-HHE, 4-HNE, DHA and EPA in thoracic aortas.
Control or fish-oil diet was fed to C57BL/6 mice for 3 weeks. The concentrations of intra-aortic 4-HHE or 4-HNE (A), and DHA or EPA (B) were measured by LC-MS/MS analyses. Each value represents the mean ± SE of 4 animals. *P<0.05, ***P<0.001, compared with each corresponding control.
Figure 3.
Effect of DHA on intracellular 4-HHE and 4-HNE levels, or Nrf2 activation in HUVECs.
(A, B) HUVECs were incubated with 75 µM DHA or EPA for 6 h. The concentrations of intracellular 4-HHE or 4-HNE were measured by LC-MS/MS analyses. Each value represents the mean ± SE of 6–8 experiments. (C) HUVECs were cotransfected with a reporter plasmid (pGL4.27(Nrf2-luc2P/minP/Hygro)) and a control plasmid (pRL-TK). After transfection, HUVECs were incubated with DHA, EPA or 4-HHE for 16 h. The ratio (reporter/control luciferase activity) obtained from control cell lysate was set at 1. Each value represents the mean ± SE of four experiments. (D, E) HUVECs were incubated with 75 µM of DHA or EPA for 6 h. (D) Nuclear lysates were subjected to western blotting analyses. Each value represents the mean ± SE of three experiments. (E) Analysis of the binding of Nrf2 in nuclear lysates to its consensus oligonucleotide was performed using the ELISA-based TransAM Nrf2 kit. Each value represents the mean ± SE of four experiments. *P<0.05, ***P<0.001, compared with each corresponding control.
Figure 4.
Effects of DHA, EPA or 4-HHE on HO-1, GCLM or p62 expression in HUVECs.
(A–D) HUVECs were incubated with DHA, EPA or 4-HHE for 6 h. The relative mRNA expressions of HO-1, GCLM, p62 or Nrf2 were quantitated using real-time RT-PCR. Each value represents the mean ± SE of 3 experiments. (E) HUVECs were incubated with DHA, EPA or 4-HHE for 6 h. Total cell lysates were subjected to western blotting analyses. (F, G) HUVECs were treated with Nrf2 siRNA or control siRNA, and incubated for 48 h. (F) The relative mRNA expression of Nrf2 was analyzed quantitatively using real-time RT-PCR. Each value represents the mean ± SE of four experiments. (G) Whole cell lysates were subjected to western blotting analyses. (H–K) HUVECs were transfected with Nrf2 siRNA or control siRNA. (H–J) After 48 h, the cells were incubated with DHA for a further 6 h. The relative mRNA expressions of HO-1, GCLM or p62 were quantitated using real-time RT-PCR. Each value represents the mean ± SE of 3 experiments. (K) 32-h after transfection, the cells were incubated with DHA (50 µM) or 4-HHE (5 µM) for a further 16 h. Whole cell lysates were subjected to western blotting analyses. *P<0.05, ***P<0.001, compared with each corresponding control, †††P<0.001, compared with the corresponding cells treated with control siRNA.
Figure 5.
Antioxidant effects of DHA in HUVECs.
(A) HUVECs were pretreated with DHA for 16 h, and then stimulated with tBHP (250 or 500 µM) for 6 h. Cell viability was determined by MTT assay. Values are expressed as percentage of cell survival, and each represents the mean ± SE of 4 experiments. (B, C) HUVECs were incubated with DHA (75 µM) for 16 h. GSH concentration (B) and ratio of GSH/GSSG (C) were determined. Each value represents the mean ± SE of 7 experiments. (D) HUVECs were pretreated with DHA (75 µM) for 16 h, and exposed to tBHP (250 or 500 µM). ROS released from cells was determined at different time intervals over 2-h period. Each value represents the mean ± SE of 8 experiments. ***P<0.001, compared with tBHP-treated BSA control, †††P<0.001, compared with tBHP-untreated BSA control.
Figure 6.
Effects of HO-1 inhibition and Nrf2 knockdown on antioxidant effects of DHA or 4-HHE.
(A) HUVECs were pretreated in the presence of DHA (75 µM) with or without ZnPP (5 µM) for 16 h, and stimulated with tBHP (250 µM) for 24 h. LDH in the supernatant of culture media was quantitatively analyzed. Each value represents the mean ± SE of 6 experiments. (B–D) HUVECs were transfected with Nrf2 siRNA and control siRNA, respectively. 32-h later, they were incubated with DHA (75 µM) or 4-HHE (5 or 10 µM) for additional 16 h, and then exposed to tBHP (250 or 500 µM). (B, C) Cell viability 6-h after tBHP treatment was measured by MTT assay. Values are expressed as percentage of cell survival, and each represents the mean ± SE of 3–4 experiments. (D) tBHP (500 µM)-induced ROS release was measured at different time intervals over 2-h period. Each value represents the mean ± SE of 4 experiments. **P<0.01, ***P<0.001, compared with tBHP-treated corresponding control, †††P<0.001, compared with tBHP-untreated BSA control.
Figure 7.
Effects of PPARα siRNA, COX inhibitors or antioxidants on DHA- or 4-HHE-induced HO-1 mRNA expression.
(A) HUVECs were treated with PPARα siRNA or control siRNA, and incubated for 48 h. The relative mRNA expression of PPARα was quantitated using real-time RT-PCR. Each value represents the mean ± SE of three experiments. (B) HUVECs were transfected with siRNA targeted against PPARα or control siRNA. After 48 h, the cells were incubated with DHA (25 µM) for a further 6 h. The relative mRNA expression was analyzed quantitatively using real-time RT-PCR. Each value represents the mean ± SE of three experiments. (C–F) HUVECs were pretreated in the presence of indomethacin (10 µM), NS-398 (1 µM), SC-58125 (1 µM), BHT (100 µM), α-tocopherol (100 µM) or NAC (5 mM) for 1 h, and stimulated with DHA (25 µM) or 4-HHE (5 µM) for 6 h. The relative mRNA expression was quantitated using real-time RT-PCR. Each value represents the mean ± SE of 3–5 experiments. **P<0.01, compared with the control cells treated with control siRNA, ††P<0.01, †††P<0.001, compared with NAC-untreated corresponding cells.
Figure 8.
A schematic figure showing the Nrf2-mediated effects of DHA on antioxidant activity and endothelial function in aortic tissue.