Naringenin promotes SDF-1/CXCR4 signaling pathway in BMSCs osteogenic differentiation

Naringenin, a dihydro-flavonoid compound that shows chemotactic activity, may have a good application prospect in repairing bone tissue, but its specific mechanism in bone regeneration, especially the osteogenic differentiation of stem cells, needs for a further study. The aim of this study was to investigate the effect of naringenin on the osteogenic differentiation and its roles in the C-X-C chemokine receptor type 4/stromal cell-derived factor 1 (SDF-1/CXCR4) signal pathway of bone marrow-derived mesenchymal stem cells (BMSCs).

BMSCs were harvested from the femurs and tibias of 4-to-6-week-old male Sprague-Dawley rats. Cell Counting kit-8 assay was used to determine cytotoxicity of naringenin. Alkaline phosphatase (ALP) activity was measured in cell's precipitates and alizarin-red staining was performed to determine the osteogenic differentiation capacity of the BMSCs. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting were adopted to determine the expression of genes and proteins.

The cellular morphology was spindle-shaped, and arranged in radial and whorled patterns. The flow cytometric analysis have confirmed the presence of characteristic surface proteins in the harvested BMSCs. Different concentrations (0-200 μg/ml) of naringenin have no influence on the viability and proliferation rate of the BMSCs. The highest ALP activity was found at culture day 7 and 9 when the concentration of naringenin was 75 and 100 μg/ml. Positive red or dark red stained cells with mineralized nodules can be observed on day 14. The expression of ALP, Runt-related transcription factor 2, CXCR4 and SDF-1a at the gene and protein levels in naringenin-treated cells were significantly higher than those in the control cells. Moreover, AMD3100, an inhibitor of CXCR4, suppressed the expression of the studied genes and proteins.

Naringenin does not show toxic effect on BMSCs. Naringenin promotes the expression of the SDF-1a gene and protein via the SDF-1/CXCR4 signaling pathway. A better understanding of the mechanisms of naringenin action would be helpful for developing specific therapeutic strategies to improve bone regeneration after injuries.