Shikonin promotes osteogenic differentiation of periodontal ligament stem cells by modulating the p38 MAPK/ETS transcription factor 1/cementum protein 1 axis in vitro.

Periodontitis is a common disorder that causes tooth loss. Osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is crucial for repairing periodontal defects. This study aimed to investigate the underlying mechanism by which Shikonin promotes the osteogenic differentiation of PDLSCs. PDLSCs were isolated from the periodontal ligaments of orthodontically extracted premolars. Flow cytometry was used to characterize the cell surface markers of PDLSCs. PDLSCs were cultured in osteogenic induction medium and treated with Shikonin. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were used to evaluate ALP activity and assess calcium nodule formation, respectively. Protein levels were determined by Western blot analysis. A dual-luciferase reporter assay and chromatin immunoprecipitation analysis confirmed the interaction between the ETS transcription factor 1 (ELK1) and cementum protein 1 (CEMP1) promoter. Shikonin promoted the osteogenic differentiation of PDLSCs (about 7.6 folds for ALP staining and about 6.9 folds for ARS staining with 1 μm Shikonin, p < 0.01). Shikonin-mediated enhancement of osteogenic differentiation was achieved by promoting the phosphorylation of ELK1 through the activation of the p38 MAPK signaling pathway (p < 0.05). Moreover, ELK1 accelerated osteogenic differentiation and transcriptionally activated CEMP1 expression (p < 0.05). Activation of the ELK1/CEMP1 axis contributed to Shikonin-induced osteogenic differentiation of PDLSCs (p < 0.05). Shikonin promotes the osteogenic differentiation of PDLSCs in vitro by modulating the p38 MAPK/ELK1/CEMP1 axis.