Figure 4

Syringaresinol elevates NO production via PLC/Ca²⁺-dependent eNOS phosphorylation. (A) HUVECs were preincubated with Fluo-4 (5 µM) for 30 minutes at 37℃ and stimulated with syringaresinol (30 µM) in the presence or absence of BAPTA-AM (5 µM). The intracellular Ca²⁺ levels were captured using a confocal laser microscope. Relative levels of intracellular Ca²⁺ were calculated from fluorescence intensities of Fluo-4. Data are the mean ± SD (n ≥ 3). ^**P < 0.01 versus control. (B-D) Cells were treated with syringaresinol (30 µM) in the presence or absence of BAPTA-AM (5 µM), U73122 (5 µM), W-7 (50 µM), Genistein (25 µM), and GF109203X (2 µM). The levels of the indicated target proteins were determined by Western blot analysis. (E) HUVECs transfected with control or CaMKKβ siRNAs (100 nM) were cultured in fresh medium for 47 h and then treated with syringaresinol for 1 h. The levels of target proteins were analyzed by Western blotting. (F) HUVECs were treated with the same conditions as above, and the intracellular levels of NO were determined by confocal microscopy. Data are the mean ± SD (n ≥ 3). ^*P < 0.05 and ^**P < 0.01 versus syringaresinol alone.