蛇床子素对大鼠信号通路AKT/eNOS/sGC/PKG及成骨细胞成熟分化的影响

Effect of osthole on AKT/eNOS/sGC/PKG signaling pathway and osteoblast differentiation and maturation of rat

目的研究蛇床子素(osthol)是否通过激活丝氨酸/苏氨酸蛋白激酶(AKT)/内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)/可溶性鸟苷酸环化酶(soluble guanylate cyclase,sGC)/蛋白激酶G(protein kinase G,PKG)信号通路促进大鼠颅骨成骨细胞(rat calvarial osteoblasts,ROBs)矿化成熟。方法检测蛇床子素处理ROBs后,成骨性转录因子RUNX2、OSX以及AKT/eNOS/sGC/PKG信号途径蛋白表达水平、免疫荧光法观察信号通路下游蛋白核易位;检测碱性磷酸酶(alkline phosphatase,ALP)活性;用AKT特异性抑制剂MK-2206阻断AKT功能后,检测MK-2206对AKT/eNOS/sGC/PKG信号途径活性及对ROBs矿化成熟的影响,进一步评估蛇床子素促进成骨细胞矿化成熟的潜在机制。结果蛇床子素处理ROBs后,成骨性转录因子RUNX2、OSX蛋白及AKT/eNOS/sGC/PKG信号通路蛋白表达量均增加,ALP活性上升;加入AKT抑制剂后,蛇床子素不再激活AKT/eNOS/sGC/PKG信号通路,阻断了蛇床子素促进ROBs的矿化成熟。结论蛇床子素可通过激活AKT/eNOS/sGC/PKG信号通路促进成骨细胞矿化成熟。

Objective To investigate whether osthol promotes mineralization and maturation of osteoblasts(ROBs)by activating AKT/endothelial nitric oxide synthase(eNOS)/soluble guanylate cyclase(sGC)/protein kinase G(PKG)signaling pathway.Methods The expression levels of osteogenic transcription factors RUNX2,OSX,and AKT/eNOS/sGC/PKG signaling pathway protein were detected after osthol treatment with rat calvarial osteoblasts(ROBs),and the downstream protein nuclear translocation of signaling pathway was observed by immunofluorescence.Alkline phosphatase(ALP)activity was detected.The function of AKT was blocked by the AKT specific inhibitor MK-2206,and the changes in the activity of AKT/eNOS/sGC/PKG signaling pathway and the effects on the mineralization and maturation of ROBs were detected.The potential mechanism of osthol promoting mineralization and maturation of osteoblasts was further evaluated.Results Osthol treated ROBs,the expression levels of osteogenic transcription factors RUNX2,OSX protein,and AKT/eNOS/sGC/PKG signaling pathway protein increased,and the activity of ALP increased.After the addition of AKT inhibitor,osthol no longer activated AKT/eNOS/sGC/PKG signaling pathway.Osthol blocked the promotion of ROBs mineralization and maturation.Conclusion Osthol can promote mineralization and maturation of osteoblasts by activating AKT/eNOS/sGC/PKG signaling pathway.