Establishment of osteoclast and osteoblast cell platforms to evaluate food factors with anti-osteoporotic potential

During menopause and aging, gradual accumulation of reactive oxygen species together with progressive decline of endogenous antioxidant defense system lead to increased oxidative stress, accelerate formation of osteoclast and promote apoptosis of osteoblast, resulting in loss of bone mass. Nuclear factor E2-related factor 2 (Nrf 2) is the key transcription factor of antioxidant defense system; studies have shown that under receptor activator of nuclear factor kappa-B ligand (RANKL) induction, the bone marrow cells isolated from Nrf 2 knockout mice exhibited higher differentiation capacity and bone resorption activity than those of wild type mice. Accordingly, we used an ARE-driven reporter stable cell line to screen Nrf2 activators, and results demonastrate that nobiletin, Chinese olive ethyl acetate extract (COE-EA), caffeamide derivative (36-25B) and genistein are our candidate compounds. Genistein is a phytoestrogen proven to reduce osteoporotic incidence. Fermentation of genistein by Bacillus subtilis natto BCRC 80517 improved water solubility and bioavailability. The bioconversion product, genistein 7-O-phosphate, following a series of purification steps was obtained with a purity of approximately 78.8%. Two in vitro osteoclast models were established from Balb/c mouse primary osteoclast culture and with the murine macrophage cell line RAW 264.7. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme of osteoclast. Investigation of the number of the TRAP-positive cells and TRAP activity was used to confirm the models. Increased ROS and decreased mRNA level of HO-1, a downstream target of Nrf 2, was observed during differentiation of the osteoclastic cells. On the other hand, such effect was abrogated by a classical Nrf 2 activator, tert-butylhydroquinone (TBHQ), suggesting that Nrf 2 activators may have anti-osteoporotic potential. In order to maintain the delicate balance between osteoclast and osteoblast in vivo, the effects of the compounds on osteoblast were observed. Primary osteoblast culture from Balb/c mouse was established andalizarin red S was used to stain calcium deposits to confirm osteoblastic mineralization. The in vitro osteoclast and osteoblast models will be useful to screen out new compounds with anti-osteoporotic potential.