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. 2015 Sep 21;7(9):8112–8126. doi: 10.3390/nu7095381

Figure 3.

Figure 3

DHA-derived 4-HHE induces Mcp-1 expression partially through the oxidative stress-induced Nrf2 pathway in VSMCs. (A,C) VSMCs were treated with N-acetyl-l-cysteine (NAC; 10 mM) for 1 h before incubation with BSA, DHA (50 μM), EPA (50 μM) or 4-HHE (25 μM) for 6 h. Relative mRNA expression of Mcp-1 (A) and Hmox-1 (C) in VSMCs was quantitated using RT-qPCR. Results were normalized against 18S rRNA and expressed as fold increase over control. (B) Reactive oxygen species (ROS) production was measured by 2′7′-Dichlorodihydrofluorescein diacetate (H2DCFDA). BSA, DHA (50 μM) or EPA (50 μM) was added for 4 h (left panel). BSA or DHA (50 μM) was added with or without NAC (10 mM) for 4 h (right panel). (D,E) VSMCs were treated with Nrf2 siRNA (40 nM) or control siRNA (40 nM). After 24 h, VSMCs were treated with vehicle or 4-HHE (25 μM) for 6 h. Relative mRNA of Nrf2 (D) and Mcp-1 (E) was quantitated using RT-qPCR. Values represent the mean ± SE of three independent experiments (n = 9; A,C); a single experiment (n = 3; B); and two independent experiments (n = 6; D,E). * P < 0.05, *** P < 0.001, compared with the corresponding control.