Sinomenine inhibited RANKL-induced NFAT, AP-1 activation.

<p>RAW264.7 cells, transiently expressing NFAT-luc or AP-1-luc reporter gene construct were pre-treated with SIN for 30 min and followed by 100 ng/ml RANKL stimulation for 12 h. (A) NFAT and (B) AP-1 transcriptions are indicated by luciferase activity. (C) RAW264.7 cells were plated into 6-well plates, incubated with RANKL (100 ng/mL) and SIN for 24 h. Real-time PCR was performed to determine gene expressions of NFATc1 and AP-1 components (c-Fos, Fra-1, Fra-2). RAW264.7 cells were incubated with SIN for 30 min and then stimulated with RANKL for 30 min. (D) c-Fos protein expression was analyzed by Western blot. β-actin was used as loading control. Densitometric quantification and statistical analysis include the results from 3 independent experiments. P<0.05; **P<0.01(compared with RANKL group); # P<0.05, # #P<0.01 (compared with control group). (E) A schematic diagram shows a hypothetical model by which SIN inhibits osteoclast differentiation and function.