Tumor necrosis factor (TNF) ? plays an important pathogenic role in the inflammatory bone resorption and accelerates osteoclastogenesis induced by receptor activator of NF?B ligand (RANKL). Epigallocatechin gallate (EGCG), one of green tea catechin components, inhibits osteoclastogenesis differentiated by RANKL. However, it is not clear an influence of EGCG for the acceleration of osteocalstogenesis by TNF? in vitro. In this study, we examined an effect of EGCG for acceleration of osteoclastogenesis by TNF? in the presence of RANKL.
Materials and Methods:
Osteoclastogenesis assay using bone marrow macrophages (BMMs) was performed in this study. BMMs from CB17 mice were stimulated at the same time by TNF? and RANKL, or by TNF? after 48h pre-stimulation with RANKL. The stimulated cells were stained with tartrate-resistant acid phosphatase (TRAP) for identification of osteoclasts. To examine the effect of EGCG to the acceleration by TNF?, EGCG were added together with TNF?. Furthermore, osteoclastogenesis inhibitory cytokines such as interleukin-4 (IL-4), interferon ? (IFN?) or osteoprotegerin (OPG) were added the culture instead of EGCG.
TNF? accelerated osteoclastogenesis induced by RANKL. The acceleration of osteoclastogenesis by TNF? after 48h pre-stimulation with RANKL was stronger than same time stimulation by TNF? and RANKL. The acceleration was strongly inhibited by addition of EGCG both condition of co-stimulation and pre-stimulation with RANKL. Osteoclastogenesis stimulated at the same time by TNF??and RANKL was clearly inhibited by IL-4, IFN? or OPG. However, the osteoclastogenesis was only slightly inhibited when those cytokines were added with TNF? after 48h pre-stimulation with RANKL.
This study indicated EGCG could inhibit osteoclastogenesis induced by RANKL and TNF?. Furthermore, EGCG could inhibit TNF?-inducing osteoclastogenesis from BMMs being 48h pre-stimulated with RANKL unlike other inhibitory cytokines. These results show usefulness of EGCG as therapeutic regents for prevention and treatment of inflammatory bone loss related with TNF.