90.    Study of the potential therapeutic role of an Argentinian plant used in antirheumatic folk medicine on bone loss induced by oestrogen deficiency

Smilax campestris Griseb. is the most widely distributed Smilax specie in the north of Argentina and it is used as antirheumatic in folk medicine. A bone-protective role of several plant polyphenols, such as flavonoids which are present in SM, was found in menopause and rheumatoid arthtritis. The aim was to analyze: a) the effect of aqueous extracts of SM (hereon SM), on the osteoclastic differentiation induced by RANKL (RL) in murine macrophages (RAW 274.4); b) the effect of SM on the viability of RL-induced OCs; c) whether the effect of SM was related to estrogenic activity of the extracts. RAW cells were cultured (5 days) with RL in the presence or absence of SM (10-1000 ng/ml) or estradiol (E2, 10-6 - 10-8 M), and the osteoclast differentiation was evaluated. We measured: multinucleated, tartrate resistant acid phosphatase (TRAP) positive (osteoclast-like) cells (histochemistry); cathepsin k mRNA expression (sq RT-PCR); matrix metalloproteinases (MMP) activity (zymography) and cell proliferation (BrdU incorporation). To analyze whether SM inhibited the translocation of NFAT transcription factor, RAW cells were preincubated with SM (1h), prior to 1 h stimulation with phorbol myristate acetate plus calcium ionophore (PIo), and then NFATc1 expression was determined (western blot). To investigate whether SM stimulated apoptosis of OC, RAW cells were cultured during 5 days with RL, and then SM or E2 was added to cultures. After 24 h, apoptosis was assessed by TUNEL assay. To analyze the role of ER, RAW cells were incubated (1h) with ERα or ERβ antagonists previous to SM treatment. Statistical analysis was applied to the results and values were considered significantly different at a p< 0.05. SM significantly diminished RL-induced osteoclast-like cells (OLCs), cathepsin k mRNA expression and MMP activity in RAW cells, in a dose dependent manner. We didn’t observe a significant decrease in the proliferation of cells treated with the extract at the concentrations employed. SM (10 ng/ml) induced apoptosis of OCs. Effect of SM on TRAP activity, NFATc1 translocation and apoptosis were reversed by the ERα antagonist. We conclude that aqueous extracts of SM are capable to significantly diminish RL-induced osteoclastic differentiation of RAW cells by decreasing cathepsin k mRNA expression, MMP activity and TRAP expression. SM also inhibits PIo induced nuclear translocation of NFAT, a master regulator of osteoclastogenesis. SM induce apoptosis on RL-induced osteoclast-like cells. Engagement of ERα is involved in the effects of SM. These mechanisms may have a role in protecting bone during menopause.