Methoxyluteolin Inhibits Neuropeptide-stimulated Proinflammatory Mediator Release via mTOR Activation from Human Mast Cells

Mast cells (MCs) are critical for allergic reactions but are also important in inflammatory processes. Stimulation by neuropeptides, such as substance P (SP) and neurotensin (NT), leads to release of preformed molecules stored in numerous MC secretory granules and newly synthesized proinflammatory mediators, including tumor necrosis factor, C-X-C motif chemokine ligand 8, and vascular endothelial growth factor. Here, we investigate the role of mammalian target of rapamycin (mTOR) signaling in the stimulation of cultured human LAD2 MCs by NT or SP, as well as the inhibitory effect of the natural flavonoids 3',4',5,7-tetrahydroxyflavone (luteolin) and its novel structural analog 3',4',5,7-tetramethoxyflavone (methoxyluteolin). Stimulation by NT (10 μM) or SP (1 μM) increases (P < 0.0001) gene expression (after 6 hours) and release (after 24 hours) of tumor necrosis factor, C-X-C motif chemokine ligand 8, and vascular endothelial growth factor. This occurs via activation of both mTOR complexes, as denoted by the increased phosphorylated (p) protein levels (P < 0.0001) of the downstream mTORC1 substrate pp70S6KThr389 and mTORC2 component pmTORSer2448. Pretreatment of human MCs using the mTORC1 inhibitor rapamycin, the mTORC1/mTORC2 inhibitor Torin1, or the two flavonoids decreases both gene expression and release (P < 0.0001) of all three mediators. Methoxyluteolin is a more potent human MC inhibitor than luteolin or Torin1, implicating other MC protein targets in addition to the mTOR complex. These findings indicate that mTOR is partially involved in the neuropeptide stimulation of MCs, but the novel flavonoid methoxyluteolin inhibits the response entirely, suggesting that it may be developed for treatment of allergic and inflammatory diseases.