Nobiletin, a dietary phytochemical, inhibits vascular smooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases pathway

Dietary flavonoids have been shown to reduce risk of cardiovascular disease, but the underlying molecular mechanisms are not known. The objective of this study was to investigate the effect of nobiletin, a dietary phytochemical belonging to polymethoxy flavonoid from the peel of Citrus fruit, on vascular smooth muscle cells (VSMCs) proliferation and its mechanisms. VSMCs proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [3H]thymidine incorporation assay. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were determined by western blotting. [Ca2+]i was measured by laser scanning confocal microscopy. Our results showed that angiotensin II-induced VSMCs proliferation was inhibited by nobiletin. While no effect on ERK1/2 and p38 MAPK, nobiletin markedly inhibited angiotensin II-induced activation of JNK. Anthra[1-9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, decreased the [3H]thymidine incorporation induced by angiotensin II. Nobiletin also attenuated both the intracellular Ca2+ mobilization and the extracellular Ca2+ influx induced by angiotensin II. Furthermore, intracellular Ca2+ chelation by BAPTA-AM, extracellular Ca2+ chelation by EGTA or blockade of L-type Ca2+ channel with verapamil inhibited angiotensin II-induced JNK activation. These findings suggest that the preventing effect of nobiletin on angiotensin II-induced VSMCs proliferation is attributed, in part, to its inhibitory effect on Ca2+-dependent JNK activation in VSMCs. Thus, inhibition of JNK by nobiletin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMCs growth.