Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation.

Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including OP and RA, with reasonable action mechanisms.