| ObjectCalpain is a calcium-dependent cysteine protease that involved in osteoclastogenesis, therefore might serve as a potential interventional target to reduce bone resorption. This study aims at investigating the mechanism of calpain on RANKL-induced osteoclastogenesis in murine RAW264.7cells, especially at whether inhibition of calpain could block the osteoclastogenesis process.Method6groups of different dosage including RANKLOng/ml,20ng/ml,50ng/ml,70ng/ml,100ng/ml and [RANKL50ng/ml+Calpastatin50nM] were applied in RAW264.7cells respectively for continuous6days to induce oscteoclastogenesis. Calpain activity was examined in5groups of RANKLO-100ng/ml on day2, and TRAP stain and pit formation assay were examined in all6groups on day6, so as to investigate whether calpain inhibition could block osteoclastogenesis. Moreover, Western Blot analysis on protein expression of NFκB and c-Fos were examined in groups that induced by RANKL50ng/ml for6h,24h,48h and by [RANKL50ng/ml+Calpastatin50nM] for48h, respectively, so as to compare the difference of protein expression exerted by Calpain inhibition.ResultCalpain activity and TRAP positive cell count as well as pit formation area were all increased in a RANKL dose-dependent manner in RAW264.7cells. Calpain inhibition using Calpastatin in the existence of RANKL significantly inhibited TRAP positive cell count and pit formation area, compared with using RANKL alone. In addition, NFκB and c-Fos protein expression were increased as the duration of RANKL induction prolonged, and were also significantly decreased after Calpastatin inhibition comparing with RANKL induction alone.ConclusionCalpain activity positively correlates with the extent of osteoclastogenesis induced by RANKL in murine RAW264.7cells. Inhibition of Calpain can effectively block osteoclastogenesis induced by RANKL. Calpain inhibition can also triggered an inhibitory effect on NFκB and c-Fos protein expression. |
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