Citrus nomilin down-regulates TNF-α-induced proliferation of aortic smooth muscle cells via apoptosis and inhibition of IκB

Nomilin is a bitter compound present in citrus and has been demonstrated as useful for various disease preventions through anti-proliferative, anti-inflammatory, and pro-apoptotic activities. Although in vitro disease models have shown that certain limonoids in the p38 mitogen-activated protein kinase signal cascade, the downstream signaling pathways remain unclear. In this study, the effects of nomilin on the proliferation and apoptotic pathways of human aortic smooth muscle cells (HASMCs) that forms the basis of progression of atherosclerotic diseases and restenosis was tested for the first time. The cellular uptake level and stability of nomilin were determined by high-performance liquid chromatography and high-resolution mass spectra. Pretreatment of HASMCs with nomilin stimulated extrinsic caspase-8, intrinsic caspase-9, and apoptotic caspase-3 and resulted in significant inhibition of TNF-alpha-induced proliferation. Additionally, results showed a decreased ratio of anti-apoptotic Bcl-2 protein to pro-apoptotic Bax (Bcl2/Bax), indicating mitochondrial dysfunction consistent with apoptosis. Furthermore, nomilin significantly decreased the phosphorylation of I kappa B alpha, an inhibitor of NF-kappa B and subsequently, reduced the downstream inflammatory signaling in TNF-alpha treated HASMCs. Our findings indicate that the anti-proliferative activity of nomilin on TNF-alpha-induced HASMCs results from apoptosis through a mitochondrial-dependent pathway and suppression of inflammatory signaling mediated through NF-kappa B.