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Endothelial nitric oxide synthase (eNOS) plays an important role in maintaining blood pressure homeostasis and vascular integrity. Natural dietary flavoniods are thought to protect against cardiovascular diseases by acting as antioxidants and vasodilatants. This study examined the effect of cyanidin-3-glucoside (Cy3G), a typical anthocyanin pigment, on eNOS expression. Treatment of bovine artery endothelial cells (BAECs) with Cy3G for 8 hours of enhanced eNOS protein expression in a dose- and time-dependent manner was determined by Western blot analysis. Longer incubation (12, 16, and 24 hours) of BAECs with 0.1 micromol/L of Cy3G caused a further increase in eNOS expression, and subsequently Cy3G also significantly increased nitric oxide output 2-fold (24 hours). Furthermore, Cy3G stimulated the phosphorylation of Src and extracellular signal-regulated kinase 1/2 (ERK1/2) in a time-dependent manner. An Src kinase inhibitor, pp2, and MEK inhibitor, PD98059, blocked the ERK1/2 phosphorylation and eNOS expression. Transfection with dominant-negative Src cDNA also inhibited the eNOS expression stimulated by Cy3G. In addition, stimulation with Cy3G for 30 minutes resulted in a phosphorylation of Sp1 that was blocked by PD98059. Cy3G enhanced the binding activity of the transcription factor Sp1 to the GC box in the proximal eNOS promoter of BAECs, as revealed by chromatin immunoprecipitation assay. The present study demonstrated that Cy3G induced eNOS expression and escalated NO production via an Src-ERK1/2-Sp1 signaling pathway in vascular endothelial cells. Increased eNOS expression may help to ameliorate endothelial dysfunction, harmonize blood pressure, and prevent atherosclerosis as long-term beneficial effects of flavoniods.

Upregulation of Endothelial Nitric Oxide Synthase by Cyanidin-3-Glucoside, a Typical Anthocyanin Pigment