Molecular mechanisms of immunomodulatory activity by polysaccharide isolated from the peels of Citrus unshiu

The extracts of the Citrus unshiu fruit induce numerous biological activities such as anticancer, anti-adipogenic, and antimicrobial effects. Furthermore, its peel has been used as a Chinese medicine for centuries and is consumed as a tea in China, Japan, and Korea. We investigated the effects of the polysaccharide isolated from C. unshiu peel (CPE-II) on cytokine and inflammatory mediator production in RAW 264.7 macrophages and performed signal transduction experiments to identify the pathways involved in its actions. CPE-II exhibited macrophage-stimulatory activity by inducing the production of interleukin (IL)-6, tumor necrosis factor (TNF)-α and nitric oxide (NO) in RAW 264.7 macrophages. Signal transduction experiments showed that CPE-II phosphorylated mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in RAW 264.7 cells in a concentration-dependent manner. The effects of CPE-II on IL-6/IL-12 and TNF-α production were completely suppressed by a specific inhibitor of c-Jun N-terminal kinase (JNK). Moreover, experiments with neutralizing antibodies showed that Toll-like receptor 2 (TLR2) and TLR4 were involved in the stimulation of IL-6 and NO production by CPE-II in RAW 264.7 cells. Collectively, these results suggest that the TLR2/4 and JNK pathways are essential for the CPE-II immune activity in RAW 264.7 cells.