The protective effect of berberine hydrochloride on LPS-induced osteoclastogenesis through inhibiting TRAF6-Ca2+-calcineurin-NFATcl signaling pathway

The present study investigated the protective effect of berberine hydrochloride on lipopolysaccharide (LPS)-induced acute bone destruction through inhibition of the TNF receptor associated factor 6 (TRAF6)-Ca2+ -calcineurin-nuclear factor of activated T-cell 1 (NFATc1) signaling pathway. An osteoclast culture system of RAW264.7 cells induced by LPS in vitro was established. A polymerase chain reaction (PCR) assay was applied to determine the effect of berberine hydrochloride on the mRNA expression levels of fos-related antigen 2 (Fra-2), tartrate-resistant acid phosphatase (TRAP), beta 3-integrin, cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), V-type proton ATPase subunit d 2 (Atp6v0d2) and NFATc1. An ELISA assay was performed to measure the release of tumor necrosis factor-alpha (TNF-alpha). Western blot analysis was used to measure the effect of berberine hydrochloride on the expression of calcineurin in the LPS-induced NFATc1 signaling pathway, as well as the expression levels of phosphoinositide phospholipase C-gamma l (PLC-gamma 1), toll like receptor 4 (TLR4) and TRAF6. The effect of berberine hydrochloride on Ca2+ concentration was detected using a confocal technique with a Flou-3/acetoxymethyl ester Ca2+ probe. The PCR results demonstrated that berberine hydrochloride inhibited the mRNA expression levels of Fra-2, TRAP, beta 3-integrin, cathepsin K, DC-STAMP, Atp6v0d2 and NFATc1. Furthermore, the ELISA results demonstrated that TNF-alpha expression was decreased. The western blot analysis revelead that berberine hydrochloride treatment results in decreased expression levels of PLC-gamma 1, TLR4 and TRAF6, and inhibition of Ca2+ influx. In conclusion, the results of the present study suggest that berberine hydrochloride targets TRAF6 and NFATc1, thus inhibiting osteoclastogenesis and bone destruction via inhibition of the TRAF6-Ca2+ -calcineurin-NFATcl signaling pathway.