Ligustilide Inhibits RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells and Its Mechanism Related by GPER

OBJECTIVE  To observe the effect of ligustilide (LIG) on the differentiation of RAW264.7 cells into osteoclasts induced by receptor activator of nuclear factor-κB ligand (RANKL), and to explore the mechanism from the perspective of G protein coupled estrogen receptor (GPER).METHODS  RAW264.7 cells were cultured in vitro and osteoclast-like cells differentiation were induced by RANKL. Osteoclast formation and differentiation were identified by tartrate resistant acid phosphatase (TRAP) staining and TRAP enzyme activity. Expressions of GPER and osteoclast differentiation related marker genes were observed by qPCR. GPER protein was assessed by Western blot. The GPER specific antagonist G36 was utilized to detect the effect of LIG on osteoclast differentiation through TRAP staining, TRAP enzyme activity, qPCR and phalloidin staining.RESULTS  TRAP enzyme activity of LIG (10 μmol/L) group was much lower than that of RANKL group (P < 0.01). TRAP staining showed that the number of TRAP positive cells of LIG group was smaller than that of RANKL group (P < 0.001). The qPCR results showed that the expressions of DC-STAMP, NFATc1, CTSK and RANK mRNA of LIG group were significantly lower than those of RANKL group (P < 0.05, P < 0.001), and the expression of GPER mRNA was significantly up-regulated (P < 0.001). Western blot showed that the expression of GPER protein of LIG group was higher than that of RANKL group (P < 0.001). After the treatment with G36, the effect of LIG on osteoclast-like cells differentiation were attenuated, the number of TRAP positive cells increased (P < 0.01), TRAP enzyme activity was enhanced (P < 0.05) and the mRNA expressions of DC-STAMP, NFATc1, CTSK and RANK significantly increased (P < 0.05).CONCLUSION  LIG can promote the expression of GPER, reduce the expression of RANK and downstream transcription factor NFATc1 in RANKL induced osteoclast, which may be one of the mechanisms involved in LIG suppressing osteoclast differentiation and bone resorption.