Objective: To establish the in vitro study model of osteoclasts induced by RANKL, to elaborate the effect of formononetin (FO) , an effective component of Caulis Spatholobi, on the differentiation and function of bone marrow mononuclear macrophages (BMMs) into osteoclasts, and to explore the molecular mechanism of its inhibition.
Methods: The BMMs in femur and tibia were isolated from 20 clean C57/BL6 mice of 4 to 6 weeks old, 10 males and 10 females, each weighing (20± 2) g. The BMMs in femur and tibia were cultured and proliferated in vitro with α-MEM medium. BMMs were cultured with MCSF and different concentrations of anthocyanin (5 to 50 μm) respectively for 4 days, and CCK8 of cell proliferation and toxicity was detected. BMMs in good growth condition were added to M-CSF and RANKL to induce osteoclast differentiation in turn. There was no special treatment in the control group. DMSO was added to the control group with DMSO solvent. Each observation group was added with different concentrations of awnasin (1 to 20 μm) . After 6 days of culture, the osteoclasts were counted and statistically analyzed. The expression of NFATc1, c-Fos and ERK, the key transcription factors in osteoclast differentiation, were detected by Western blot, RNA was extracted at 4 days, and the activity of ctsk, trap, MMP9 and Car2 were detected by real time PCR.
Results: CCK8 test results showed that awnstein could inhibit the activity of BMMs in a dose-dependent manner, and had no significant toxic effect on the growth of bmms within the safe concentration range of ≤20 μM (P= 0.278>0.05) . The results of trap staining showed that awnstein could inhibit osteoclast production in a dose-dependent manner in the concentration range of (1 to 20 μM) , especially in 10 μM (P=0.000<0.05) . Western blot showed that 10 μ m could significantly inhibit the expression of NFATc1 and c-Fos, but not the expression of ERK. In terms of osteoclast function, the expression of ctsk (P=0.000<0.05) , trap (P=0.000<0.05) , MMP9 (P=0.000<0.05) and Car2 (P=0.000<0.05) related to osteoclast function were detected by real time PCR.
Conclusion: The effective component of Caulis Spatholobi can inhibit the proliferation and differentiation of primary mononuclear macrophages into osteoclasts, and down regulate the expression of osteoclast bone resorption related proteins and genes, which may be one of the mechanisms of its prevention and treatment of bone destruction and collapse in osteonecrosis of femoral head.
Keywords: Bone marrow macrophages; Formononetin; Osteoclastogenesis; Tartrate resistant acid phosphatase.