Notoginsenoside R1 Promotes Osteogenic Differentiation of Dental Pulp Stem Cells via MAPK Pathway

Abstract

Background: Notoginsenoside R1 (NGR1) is a bioactive compound of Panax notoginseng (Burk.) F.H. Chen (PNS), which possesses desirable properties in bone fracture healing and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Whether NGR1 can promote osteogenic differentiation of human dental pulp stem cells (DPSCs) is still unknown. This study aimed to assess the biocompatibility of NGR1 and its impact on DPSCs.

Methods: DPSCs were obtained from human wisdom teeth. Flow cytometry and multilineage differentiation were applied to determine stem cell properties. Then, the cells were treated with NGR1 for 1, 2 and 3 days, and its efficacy was detected by means of a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Alizarin red staining (ARS), alkaline phosphatase (ALP) activity, quantitative calcium node analysis, western blot and reverse-transcription-quantitative polymerase chain reaction (RT-qPCR) were executed to detect osteogenic differentiation-related proteins and genes. Western blot was also performed to assess the activation levels of the p38 mitogen-activated protein kinase (p-38 MAPK), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and extracellular signal-regulated protein kinase mitogen-activated protein kinase (ERK MAPK) pathways in DPSCs following treatment with NGR1.

Results: DPSCs were positive for CD105 and CD166, while negative for CD34 and CD45. NGR1 at concentrations of 10 and 100 μg/mL did not exhibit cytotoxicity (p > 0.05), the group of cells receiving 200 μg/mL and 500 μg/mL NGR1 exhibited proliferation inhibition on the second day as well as on the third day (p < 0.05). Compared to the control group (no treatment), the cells treated with 100 μg/mL NGR1 exhibited significantly higher ALP expression and calcium deposition. The 100 μg/mL NGR1 group also showed higher expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Collagen Type I (COL-1), and Osteocalcin (OCN) at both protein and gene levels. Western blot analysis revealed that NGR1 activated the MAPK pathway by upregulating p38 and ERK, but not JNK, in DPSCs. When the p38 and ERK signaling pathways were inhibited by SB203580 and U0126, the gene expression levels of OSX, RUNX2, COL-1, and OCN were significantly decreased (p < 0.05), but such alterations were not observed with the inhibition of the JNK pathway.

Conclusion: At the concentration of 100 μg/mL, NGR1 enhances DPSC osteogenic differentiation by regulating the MAPK pathways.

Keywords: dental pulp stem cells; mineralization; notoginsenoside R1; osteogenesis.