Celastrol By Inhibiting Chemokine CCL4Reduce Osteoclast Differentiation And Migration

Objective:To study the expression of the chemokine family in osteoclast differentiation and the effect of Celastrol on CCL4pathway in inhibiting osteoclast differentiation and osteoclast precursors migration.Methods:1. The expression of the chemokine family in osteoclast differentiationRat bone marrow was stimulated with M-CSF and RANKL, after72h the expression of factor chemokine family were assayed by gene chips and validated results by real-time PCR. Osteoclast formation was evaluated by counting positive cells for tartrate-resistant acid phosphatase (TRAP) staining. Quantitative gene expression analysis for osteoclastic markers was performed by Real time quantitative PCR.2.The expression of CCL4in osteoclast differentiation and migration.The expression of CCL4in osteoclast differentiation was detected by PCR. RAW264.7cells treatment with CCL4-neutralizing antibody to Observat the migration of osteoclast precursors and osteoclasts. The transwell chambers were used for detecting the chemotactic migration of CCL4-neutralizing antibody on RAW264.7cells.3.The effect of Celastrol on osteoclast differentiation and osteoclast precursors migration Generation and activity of Rat bone marrow effected by Cel were measured by CCK-8, then using safe concentration Cel effect Osteoclast for3d, the mRNA were measured by PCR analysis for relative expression of CCL4. The effect of Celastrol on osteoclast differentiation was evaluated by counting positive cells for tartrate-resistant acid phosphatase (TRAP) staining.The transwell chambers were used for detecting the chemotactic migration of Celastrol on RAW264.7cells.Results:1. The whole genome oligonucleotide microarray Was applied to evaluate the gene expression profile of Bone marrow-derived osteoclasts. The expressions of CCL7、 CCL4、CCL12in bone marrow-derived osteoclasts were significantly increased more than the other in chemokine family.2. Celastrol (0.1-0.5μmol/L) could inhibit the differentiation of rat bone marrow cells in adose-dependent manner and reduced expression of CCL4.RAW264.7expressed RANK. RANKL increased themigration of RAW264.7and Celastrol inhibited the cells migration. CCL4-neutralizing antibody blocked the expression of CCL4and inhibited the osteoclast precursors migration.Conclusion:1. The expressions of CCL7、CCL4、CCL12in osteoclasts differentiation were significantly increased. And CCL4plays an important role in osteoclast differentiation and osteoclast precursors migration.2. Cel could inhibite osteoclast differentiation and osteoclast precursors migration, the possible mechanism is adjusting the expression of CCL4.