Multiple sclerosis is an organ-specific autoimmune disease that targets the myelin antigen in the central nervous system. Nobiletin is a dietary polymethoxylated flavonoid found in citrus fruits. In this study, we investigated how nobiletin affects the disease state and immune responses to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis mice. Nobiletin was administered orally from 14 days before immunization until the end of the experiment, and clinical scores were determined. The production levels of interleukin-17A and interferon-γ were measured in a culture supernatant of splenocytes stimulated with myelin oligodendrocyte glycoprotein. In addition, flow cytometric analysis was performed to examine the effect of nobiletin on T cell differentiation in vitro . Admin­istration of nobiletin significantly decreased the clinical score and interleukin-17A production in splenocytes. Furthermore, in vitro analysis showed that nobiletin significantly suppressed Th17 cell differentiation and interleukin-17A production in a dose-dependent manner. The results suggest that nobiletin attenuates experimental autoimmune encephalomyelitis severity through modulation of Th17 cell differentiation.

Regents NOB, NTD and HMF were provided by Ushio-Chemix Co. (Shizuoka, Japan). Ovalubumin (OVA) 323-339 peptide was synthesized by Sigma-Aldrich (MO, USA) and the purity was over 97%. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from Tokyo Chemical Ind. Co. Ltd. (Tokyo, Japan). Mice BALB/c mice (Japan SLC, Shizuoka, Japan) and DO11.10 mice on a BALB/c background (The Jackson Laboratory, ME, USA) were maintained under specific pathogenfree conditions with a 12-h light:dark cycle at 25 ± 2°C and 55 ± 10% relative humidity. The mice were given free access to water and food throughout the experiment. All studies were performed in accordance with the ethical guidelines for animal experimentation by the Institution of Biomedical Sciences, The University of Tokushima, Japan and were approved by the institution review board of the animal ethics committee. Proliferation Assay Splenocytes (5 x 10 5 cells/well) from DO.11.10 mice were treated with NOB, NTD or HMF for 24 h in 96-well flat-bottom plate and then further cultured with 5 µg/mL OVA 323-339 peptide for 48 h at 37°C in a total volume 100 μL. Twenty μL of MTT solution was added to the culture 4 h before the end of culture. Fifty μL of 10% SDS solution was added to the well and incubated overnight at 37°C. Absorbance at 550/630 nm was measured using a microplate reader. Cytokine Production Splenocytes (2.5×10 6 cells/well) from DO11.10 mice were pretreated with NOB, NTD or HMF in a 48-well flat-bottom plate at 37°C under 5% CO 2 for 24 h and then the cells were stimulated with 5 µg/mL OVA OVA 323-339 peptide for 48 h. After the culture, culture superna

We examined the effects of polymethoxyflavonoids (PMFs) on T helper (Th) 17 cell differentiation in vitro and in vivo. Five different PMFs including nobiletin (NOB), sudachitin (SUD), demethoxysudachitin, heptamethoxyflavone and natsudaidain were used for the in vitro study, and effects of those flavonoids on Th17 responses were investigated. NOB and heptamethoxyflavone significantly suppressed the proliferation response, but SUD, demethoxysudachitin and natsudaidain did not suppress the proliferation response. All of the five flavonoids decreased IL-17A production. Mice with experimentally induced autoimmune encephalomyelitis were used as an in vivo Th17 differentiation model. We focused on two flavonoids, NOB and SUD, and examined the effects of those flavonoids. NOB significantly suppressed Th17 cell proliferation and cytokine responses, but SUD only decreased proliferation responses. The results suggest that the suppressive effect of NOB on Th17 response in vivo is stronger than that of SUD.