Shikonin intervenes with osteoclast formation and osteoclast-related gene expression
Bo Yujia1, 2, Lin Jing1, 2, Wang Liping1, 2, Zhao Jin1, 2
1Department of Dentistry and Endodontics, First Affiliated Hospital (Affiliated Stomatological Hospital) of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China; 2Institute of Stomatology of Xinjiang Uygur Autonomous Region, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
Abstract: BACKGROUND: The activation of osteoclast plays an important role in the occurrence and development of periodontitis. In recent years, shikonin has been used as an adjuvant drug after periodontal non-surgical treatment because of its small side effects and anti-inflammatory properties.
OBJECTIVE: To discuss the effects of shikonin on osteoclast formation induced by receptor activator for nuclear factor-κB ligand (RANKL) and osteoclast-related gene expression.
METHODS: The toxicity of different concentrations of shikonin (0, 0.062 5, 0.125, 0.25, 0.5, 1, and 2 μmol/L) to RAW264.7 cells was detected by cell counting kit-8 assay, and the optimal concentration was determined. RAW264.7 cells were induced into osteoclasts by different concentrations of RANKL (0, 30, 50, 100 μg/L). The number of osteoclasts was revealed by tartrate resistant acid phosphatase staining, and the optimal concentration of RANKL was determined. After treatment with different concentrations of shikonin, the effects of RANKL on osteoclast formation were detected by tartrate resistant acid phosphatase staining and tartrate resistant acid phosphatase activity. The mRNA expressions of osteoclast marker genes, including matrix metalloproteinase 9, cathepsin K, receptor activator for nuclear factor-κB, nuclear factor of activated T cells and proto-oncogene c-Fos, were detected by real-time quantitative PCR.
RESULTS AND CONCLUSION: Shikonin at a concentration of higher than 0.5 μmol/L significantly inhibited the growth of RAW264.7 cells (P < 0.05). The optimal induction concentration of RANKL was 50 μg/L. Shikonin inhibited osteoclast formation in a concentration-dependent manner (P < 0.05). Shikonin also inhibited the expression of matrix metalloproteinase 9, cathepsin K, receptor activator for nuclear factor-κB, nuclear factor of activated T cells, and proto-oncogene c-Fos in a concentration-dependent manner (P < 0.05). To conclude, shikonin can inhibit RANKL-induced osteoclast formation by inhibiting the expression of osteoclast-related genes in a concentration-dependent manner in vitro.
Key words: shikonin, osteoclast formation, periodontitis, RAW264.7, receptor activator for nuclear factor-κB ligand, induction, nuclear factor of activated T cells, proto-oncogene c-Fos