Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation

Aim: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions.

Methods: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The Apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of Caspase-3 in osteoclasts was measured using Western blotting and the Caspase-3 activity colorimetric method.

Results: SIN (0.25-2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the Apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the Caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of Caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced Caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO.

Conclusion: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated Apoptosis, thus it is a potential agent for treating excessive bone resorption diseases.