苔黑酚葡萄糖苷通过抑制糖皮质激素受体入核保护地塞米松诱导成骨细胞损伤作用  

The protective effect of orcinol glucoside on dexamethasone induced osteoblast cell damage through inhibition of glucocorticoid receptor nucleus translocation

作　　者：何心芸溪 赵婉璐 俞阳 刘燕 张巧艳 张泉龙 秦路平 HE Xin-Yun-xi;ZHAO Wan-lu;YU Yang;LIU Yan;ZHANG Qiao-yan;ZHANG Quan-long;QIN Lu-ping(School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学药学院,浙江杭州310053

出　　处：《中国药理学通报》2023年第7期1346-1353,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No.81973534);浙江省基础公益研究计划(No.LY19H280002)。

摘　　要：目的 考察苔黑酚葡萄糖苷对地塞米松(dexamethasone,DEX)诱导成骨细胞损伤的保护作用,并探讨其调控机制。方法 从新生小鼠颅盖骨提取并培养原代成骨细胞(osteoblast,OB),用DEX(1μmol·L^(-1))诱导成骨细胞损伤为模型。采用CCK-8法测定不同浓度苔黑酚葡萄糖苷及有无DEX干预下对成骨细胞增殖的影响;采用试剂盒进行成骨细胞碱性磷酸酶(ALP)染色和ALP活性测定;采用茜素红S染色观察骨矿化结节的形成;采用Annexin V-FITC法检测细胞凋亡情况;采用免疫荧光结合激光共聚焦显微镜观察细胞内糖皮质激素受体(glucocorticoid receptor,GR)的入核情况;采用Western blot法检测GR在细胞核、细胞质和全细胞中的蛋白表达,以及细胞中成骨特征蛋白CollagenⅠ、Runx2、Osterix(Osx)和Dlx5的表达。结果 与对照组相比,DEX组成骨细胞增殖、分化和矿化均明显降低(P<0.05、0.01),细胞凋亡明显升高(P<0.01);与模型组相比,苔黑酚葡萄糖苷组成骨细胞增殖、分化和矿化均明显升高(P<0.01),细胞凋亡明显降低(P<0.05)。与对照组相比,DEX组成骨细胞中GR在核内的表达明显升高(P<0.01),而GR在全细胞和细胞质中的表达明显降低(P<0.01),同时成骨特征蛋白表达也明显降低(P<0.01);与模型组相比,苔黑酚葡萄糖苷组GR在成骨细胞核内的表达明显降低(P<0.01),而GR在全细胞和细胞质中的表达明显升高(P<0.01),成骨特征蛋白的表达均明显升高(P<0.01),且呈剂量依赖关系。结论 苔黑酚葡萄糖苷能够改善DEX诱导的成骨细胞增殖、分化和矿化的抑制作用,并减少其凋亡,其作用机制与苔黑酚葡萄糖苷抑制GR入核,从而调控成骨相关蛋白表达有关。Aim To investigate the protective effect of orcinol glucoside on dexamethasone(DEX)-induced osteoblast injury and its mechanism.Methods Primary osteoblasts were extracted from calvaria of neonatal mice and cultured in medium with DEX(1μmol·L^(-1))and different concentrations of OG.CCK-8 method was used to determine the osteoblast proliferation.Alkaline phosphatase(ALP)staining and its activity were used by kit to measure the osteoblasts differenctiation.Alizarin red S staining was used to observe the formation of bone mineralized nodules.Annexin V-FITC was used to detect the apoptosis of cells.Immunofluorescence combined with laser confocal microscope was used to observe the nucleation of glucocorticoid receptor(GR)in cells.Western blot was used to detect the expression of GR in nucleus,cytoplasm and whole cells,as well as the expression of osteogenic characteristic proteins CollagenⅠ,Runx2,Osx and Dlx5 in cells.Results Compared with the control group,the proliferation,differentiation and mineralization of osteoblast(OB)in DEX groups were significantly reduced(P<0.05,0.01),and the apoptosis rate was significantly raised(P<0.01).Compared with DEX group,the proliferation,differentiation and mineralization of OB in the OG groups with different concentrations significantly increased(P<0.01),and the apoptosis rate was significantly reduced(P<0.05).Compared with the control group,in the DEX group the expression of GR in the nucleus increased significantly(P<0.01),while the expression of GR in cells and cytoplasm decreased significantly(P<0.01),and the expression of osteogenic characteristic proteins also decreased significantly(P<0.01).Compared with DEX group,the expression of GR in the nucleus decreased significantly(P<0.01),in the OG groups with different concentrations,while the expression of GR in the whole cell and cytoplasm increased significantly(P<0.01),and the expression of osteogenic characteristic proteins increased significantly(P<0.01)in a dose-dependent manner.Conclusions OG could improve the inhibiti

关 键 词：仙茅 苔黑酚葡萄糖苷 地塞米松 成骨细胞 糖皮质激素 糖皮质激素受体 

分 类 号：R-332[医药卫生] R284.1R329.24R329.25R977.11