Department of Orthodontics, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R., China.
Oral and Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou, China.
Folia Histochem Cytobiol. 2021;59(1):66-73. doi: 10.5603/FHC.a2021.0008. Epub 2021 Mar 11.
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.
柚皮素是一种二氢黄酮类化合物，具有趋化活性，在修复骨组织方面可能具有良好的应用前景，但它在骨再生中的具体机制，特别是对干细胞的成骨分化，需要进一步研究。本研究旨在探讨柚皮素对骨髓间充质干细胞（BMSCs）成骨分化的影响及其在 C-X-C 趋化因子受体 4/基质细胞衍生因子 1（SDF-1/CXCR4）信号通路中的作用。
从 4-6 周龄雄性 Sprague-Dawley 大鼠的股骨和胫骨中分离 BMSCs。采用细胞计数试剂盒-8 法测定柚皮素的细胞毒性。测定细胞沉淀中的碱性磷酸酶（ALP）活性，并进行茜素红染色，以确定 BMSCs 的成骨分化能力。采用实时聚合酶链反应、酶联免疫吸附试验和蛋白质印迹法测定基因和蛋白的表达。
细胞形态呈梭形，呈放射状和漩涡状排列。流式细胞术分析证实了收获的 BMSCs 存在特征性表面蛋白。不同浓度（0-200μg/ml）的柚皮素对 BMSCs 的活力和增殖率没有影响。当柚皮素浓度分别为 75 和 100μg/ml 时，第 7 天和第 9 天 ALP 活性最高。第 14 天可见矿化结节阳性的红色或暗红色染色细胞。与对照组相比，柚皮素处理细胞中 ALP、Runt 相关转录因子 2、CXCR4 和 SDF-1a 的基因和蛋白表达均明显升高。此外，CXCR4 抑制剂 AMD3100 抑制了研究基因和蛋白的表达。
柚皮素对 BMSCs 无毒性作用。柚皮素通过 SDF-1/CXCR4 信号通路促进 SDF-1a 基因和蛋白的表达。深入了解柚皮素作用的机制将有助于开发特定的治疗策略，以改善损伤后骨再生。
