Effect of mechano-growth factor on osteoclast activity and its mechanism / 中华创伤杂志

ABSTRACT

Objective:To investigate the effect of mechano-growth factor（MGF）on osteoclast activity and its mechanism.Methods:The RAW264.7 precursor osteoclast cell line was cultured with 25 ng/ml macrophage-colony stimulating factor（M-CSF）and 30 ng/ml receptor activator of NF-κB ligand（RANKL），and identified by tartrate resistant acid phosphatase（TRAP）staining after 7 days of culture. Western blot anslysis was used to determine the effect of 45 ng/ml MGF on the phosphoinositide-3-kinase/protein kinase B（PI3K/AKT）signaling pathway in separated osteoclasts，including levels of AKT，phosphorylation（p）-AKT，lactation mammalian target of rapamycin（mTOR），p-mTOR and TRAP at 0，4，8 and 12 hours. Real-time fluorescence quantitative PCR was used to expressions of TRAP in osteoclasts at 0，4，8 and 12 hours. The PI3K/Akt phosphorylation inhibitor LY294002（20 μmol/L）combined with MGF（45 ng/ml）was used to act on osteoclasts，and expression levels of Akt，p-Akt，mTOR，p-mTOR and TRAP were detected by Western blot at 0，4，8 and 12 hours.Results:After culturing RAW264.7 cells with M-CSF and RANKL for 7 days，a large number of osteoclasts with positive TRAP staining can be obtained. Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-Akt and p-mTOR increased continuously from（2.18±0.34）pg/ml and（0.83±0.10）pg/ml at 0 hour to（3.86±0.36）pg/ml and（1.56±0.19）pg/ml at 12 hours（ P<0.05），and expression level of TRAP decreased significantly over time，from（5.66±0.47）pg/ml at 0 hour to（3.76±0.38）pg/ml at 12 hours（ P<0.05）. Real-time fluorescence quantitative PCR analysis of expression of TRAP in osteoclasts showed that MGF inhibited the expression of TRAP in osteoclasts，which decreased from 1.02±0.06 at 0 hour to 0.53±0.11 at 12 hours（ P<0.05）. After acting LY294002 combined with MGF on osteoclasts，Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time（ P>0.05），expression levels of p-AKT and p-mTOR decreased significantly from（3.28±0.18）pg/ml and（3.29±0.22）pg/ml at 0 hour to（2.06±0.34）pg/ml and（2.04±0.20）pg/ml at 12 hours（ P<0.05），and expression level of TRAP had no significant difference over time（ P>0.05）. Conclusions:MGF inhibits osteoclast activity by inhibiting the expression of TRAP in osteoclasts through PI3K/Akt signaling pathway. LY294002 inhibits the expression of PI3K/Akt signaling pathway in osteoclasts，further verifying the mechanism of MGF inhibiting osteoclast activity，and this finding puts forward new ideas for clinical prevention and treatment of osteoporosis.