Shikonin stimulates MC3T3-E1 cell proliferation and differentiation via the BMP-2/Smad5 signal transduction pathway

Shikonin, the predominant naphthoquinone pigment isolated from the Chinese plant Lithospermum erythrorhizon, is anti-inflammatory, antiviral and exerts anticancer effects, amongst other biological activities. However, it is unknown whether shikonin affects bone formation. In the present study, the role of shikonin on cell proliferation was assessed via MTT assay, and shikonin was identified to markedly promote cell growth in a time-and dose-dependent manner in the MC3T3-E1 cell line. In addition, flow cytometric analysis was performed to evaluate the effect of shikonin on the cell cycle, and it was observed that shikonin markedly increased the percentage of S-phase MC3T3-E1 cells to accelerate the G(1)/S transition. To investigate the potential molecular mechanism by which shikonin enhances bone formation, the changes in bone morphogenic protein-2 (BMP-2), SMAD family member 5 (Smad5), runt related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) expression levels induced by shikonin were investigated using western blot analysis and quantitative polymerase chain reaction. The results indicated that shikonin increased the BMP-2 and Smad5 mRNA levels, and upregulated Smad5 and Runx2 protein expression levels to promote osteoblast differentiation. Furthermore, ALP staining was performed, and revealed that shikonin enhanced ALP activity. These results indicate that shikonin promotes cell proliferation and differentiation of MC3T3-E1 cells via the BMP-2/Smad5 signaling pathway.