Effects of HO-1 inhibition and Nrf2 knockdown on antioxidant effects of DHA or 4-HHE.

<p>(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. **<i>P</i><0.01, ***<i>P</i><0.001, compared with tBHP-treated corresponding control, <b>^†††</b><i>P</i><0.001, compared with tBHP-untreated BSA control.</p

Antioxidant effects of DHA in HUVECs.

<p>(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. ***<i>P</i><0.001, compared with tBHP-treated BSA control, <b>^†††</b><i>P</i><0.001, compared with tBHP-untreated BSA control.</p

Effects of fish-oil diet on HO-1 expression and endothelium-dependent vasorelaxation in thoracic aortas.

<p>Control or fish-oil diet was fed to C57BL/6 or Nrf2<i>^−/−</i> 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<i>^−/−</i> mice (D, F) fed with fish-oil diet for 3 weeks. Each value represents the mean ± SE of 12–18 rings. *<i>P</i><0.05, **<i>P</i><0.01, compared with control diet-fed C57BL/6 mice. <b>^††</b><i>P</i><0.01, compared with fish-oil diet-fed C57BL/6 mice.</p

Fatty acid composition (wt%) of dietary fats.

<p>Fatty acid composition (wt%) of dietary fats.</p

Effects of fish-oil diet on concentrations of 4-HHE, 4-HNE, DHA and EPA in thoracic aortas.

<p>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. *<i>P</i><0.05, ***<i>P</i><0.001, compared with each corresponding control.</p

Composition of the experimental diets.

<p>Composition of the experimental diets.</p

A schematic figure showing the Nrf2-mediated effects of DHA on antioxidant activity and endothelial function in aortic tissue.

<p>A schematic figure showing the Nrf2-mediated effects of DHA on antioxidant activity and endothelial function in aortic tissue.</p

Effects of PPARα siRNA, COX inhibitors or antioxidants on DHA- or 4-HHE-induced HO-1 mRNA expression.

<p>(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. **<i>P</i><0.01, compared with the control cells treated with control siRNA, <b>^††</b><i>P</i><0.01, <b>^†††</b><i>P</i><0.001, compared with NAC-untreated corresponding cells.</p