Citrus unshiu flower inhibits LPS-induced iNOS and COX-2 via MAPKs in RAW 264.7 macrophages

In the present study, we investigated the effects of Citrus unshiu flower on regulatory mechanisms of cytokines and nitric oxide (NO) involved in immunological activity of RAW 264.7 macrophages. Our results indicated that ethyl acetate fraction of Citrus unshiu flower (CUF-EA) downregulated LPS-induced nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, thereby reducing the production of NO and prostaglandin E-2 (PGE(2)) in LPS-activated RAW 264.7 macrophages. Furthermore, CUF-EA suppressed LPS-induced production of proinflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF)-a. To elucidate its anti-inflammatory mechanisms, CUF-EA was investigated as an inhibitor of phosphorylation of mitogen-activated protein (MAP) kinase in LPS-stimulated RAW 264.7 macrophages. As expected, the phosphorylation of MAP kinases (p38, ERK1/2 and JNK) in LPS-stimulated RAW 264.7 macrophages was suppressed by CUF-EA in a dose-dependent manner. These results suggest that the anti-inflammatory properties of CUF-EA might results from inhibition of NO, PGE(2), iNOS, COX-2, IL-6 and TNF-a expressions through the down-regulation of phosphorylation of MAPKs in RAW 264.7 macrophages.