Molecular mechanism of the combination of mechanical strain stimulation and icariin on inhibiting the differentiation of osteoclasts induced by fatigue load stimulation

Abstract

Objective To investigate the effect and molecular mechanism of the combination of mechanical strain stimulation and icariin (ICA) on inhibiting the differentiation of osteoclasts induced by fatigue load stimulation. Methods The mouse mononuclear macrophage cell line RAW264.7 was cultured in vitro, and the blank control group was α-MEM complete medium. In the fatigue load group, RAW264.7 cells were treated with 5 000 μe mechanical stretch strain, and then cultured in an osteoclast culture medium that was an α-MEM complete medium containing 40 ng/ml macrophage colony-stimulating factor and 40 ng/ml osteoclast differentiation factor. In the mechanical stimulation + ICA group, RAW264.7 cells were treated as the same procedure in the fatigue load group, and then cultured in an α-MEM complete medium containing 1×10-5 mol/L ICA simultaneously with a 1000 μe tensile strain on the substrate. The activity of tartrate-resistant acid phosphatase (TRAP) was detected using a TRAP assay kit. The mRNA expression of the osteoclast marker genes, i.e. TRAP, cathepsin K(CTSK) and matrix metalloproteinase 9 (MMP-9) was detected by real-time RT-PCR. The nuclear translocation of nuclear factor kappa B (NF-κB) was analyzed by Western Blot. Results Compared with the fatigue load group, the combination of mechanical stimulation (1 000 μe substrate stretching) and ICA (1×10-5 mol/L) could significantly inhibit the activity of TRAP in osteoclasts (P<0.01) and reduce osteoclastosis. Moreover, that combination not only could down-regulate the mRNA expression of TRAP, CTSK and MMP-9 and the differences were statistically significant (all P<0.01), but also could inhibit the formation of osteoclasts by inhibiting the phosphorylation of P65, P50 and IκB-α in NF-κB signaling pathway. Conclusions The coupling of mechanical stimulation and ICA can effectively inhibit the osteoclast differentiation and the bone resorption induced by fatigue load, and the mechanism may involve regulating NF-κB signaling pathway. Key words: Mechanical strain stimulation; Icariin; Osteoclasts; Nuclear factor-kappa B