Inhibitory effect of 8-prenylnaringenin on osteoclastogensis of bone marrow cells and bone resorption activity

This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts.  Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in α-MEM containing 10% FBS.  8-PNG was added into culture media at 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ mol·L⁻¹, separately.  17β-Estradiol (E2, 1×10⁻⁷ mol·L⁻¹) was used as positive control.  TRAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days.  Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately.  The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR.  8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG- supplemented groups.  The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly.  All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1×10⁻⁵ mol·L⁻¹ 8-PNG.  8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation.