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脉冲电磁场(pulse electromagnetic fields,PEMFs)能够促进大鼠成骨细胞(rat osteoblast cells,ROB)成熟矿化,但是其作用机制并不明确。本实验主要研究PEMFs促进大鼠成骨细胞成熟矿化与NO/c GMP信号途径的关系,进而阐明PEMFs促进成骨细胞成熟矿化的机理。首先,将成骨细胞经50 Hz、0.6 m T脉冲电磁场作用不同时间后,检测细胞培养液中一氧化氮(nitric oxide,NO)和细胞内3’-5’-环鸟苷一磷酸(3’-5’-cyclic-GMP,c GMP)的含量,以探明电磁场是否影响NO和c GMP的合成;其次,应用蛋白质印迹,检测细胞内e NOS、i NOS和PKG-1的蛋白表达量;最后,利用NOS的阻断剂L-NAME抑制NO信号通路后,检测成骨性相关指标,包括碱性磷酸酶(ALP)活性、钙化结节数量、成骨性基因Bmp-2、Collagen-1、Osterix及破骨细胞调节因子Rankl基因的表达量。结果发现,经PEMFs处理后,NO含量及c GMP含量均有明显升高;细胞内e NOS、i NOS和PKG-1蛋白表达量较空白对照组均有显著升高,说明PEMFs能够激活NO/c GMP信号途径。且经PEMFs处理的成骨细胞,ALP活性升高,BMP-2、Collagen-1和Osterix基因表达量显著增加,Rankl基因表达量下降,成骨细胞形成钙化结节的能力增强。当加入L-NAME,PEMFs引起的ALP活性增加、成骨性基因表达升高和钙化结节形成能力增强的趋势均被显著抑制。上述结果表明,经PEMFs处理成骨细胞成熟矿化过程中,NO/c GMP信号通路被激活;如该通路被抑制,则电磁场促成骨作用被抵消,说明脉冲电磁场促进成骨细胞成熟矿化依赖于NO/c GMP信号通路。
Pulsed electromagnetic fields (PEMFs) promote the mineralization of rat osteoblasts (ROB), but their mechanism of action is not clear. In this study, we mainly studied the relationship between PEMFs promoted the mineralization of osteoblasts and NO / c GMP signaling pathway in order to elucidate the mechanism by which PEMFs promote the mineralization of osteoblasts. First, the osteoblasts were treated with 50 Hz, 0.6 m T pulsed electromagnetic fields for different time to detect the nitric oxide (NO) and 3’-5’-cyclic guanosine monophosphate (3 ’ 5’-cyclic-GMP, c GMP) in order to explore whether the electromagnetic field affects the synthesis of NO and c GMP. Secondly, Western blotting was used to detect the protein expression of eNOS, iNOS and PKG- Finally, the NO-signaling pathway was blocked by L-NAME, a blocker of NOS. Osteogenic parameters such as alkaline phosphatase (ALP) activity, number of calcified nodules, Bmp-2, Collagen- , Osterix and osteoclast regulatory factor Rankl gene expression levels. The results showed that after treatment with PEMFs, NO content and cGMP content were significantly increased; intracellular eNOS, iNOS and PKG-1 protein expression levels were significantly higher than the control group, indicating that PEMFs can activate NO / c GMP signaling pathway. The osteoblasts treated with PEMFs had higher ALP activity, significantly increased the expression of BMP-2, Collagen-1 and Osterix, decreased the expression of Rankl, and enhanced the ability of osteoblasts to form calcified nodules. When L-NAME was added, PEMFs-induced increase in ALP activity, osteogenic gene expression and calcification nodule formation capacity were significantly inhibited. The above results indicated that the NO / c GMP signaling pathway was activated during the mineralization of PEMFs-treated osteoblasts, and if the pathway was inhibited, the bone-forming effect of the electromagnetic field was offset, indicating that pulsed electromagnetic fields promoted osteoblast maturation and mineralization NO / c GMP signaling pathway.