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

Figure 2.

Figure 2

DHA, EPA, and 4-HHE induce Mcp-1 expression through the p38 mitogen-activated protein kinase (MAPK) pathway in VSMCs. VSMCs (Passage 4–12) were treated with the indicated reagent for 6 h (A). (B) Primary vessels and vascular smooth muscle cells (VSMCs) (Passage 1) were treated with BSA, DHA (50 μM), EPA (50 μM) or 4-HHE (25 μM) for 6 h. Relative mRNA expression of Mcp-1 was quantitated using RT-qPCR. The results were normalized against 18S rRNA and expressed as fold increase over control. (C) 4-HHE and 4-HNE content in VSMCs were measured using LC-MS/MS. (D) p38, ERK, JNK and their phosphorylated forms, and β-actin were determined by Western blotting. DHA (50 μM), EPA (50 μM) or 4-HHE (25 μM) were added for 10 min. (E) Pretreatment with p38 kinase inhibitor (SB203580; 10 μM), ERK inhibitor (PD98059; 25 μM) or JNK inhibitor (SP600125; 10 μM) was performed for 30 min before BSA, DHA, EPA or 4-HHE incubation. The results were normalized against 18S rRNA and expressed as fold increase over corresponding control. (A) Values represent the mean ± SE of four independent experiments (n = 9); (B) a single experiment (n = 3); (C) a single experiment (n = 3); or (E) three independent experiments (n = 3–9). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with corresponding control.