南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (11): 1892-1900.doi: 10.12122/j.issn.1673-4254.2023.11.09

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冬凌草甲素逆转硫代乙酰胺对破骨和成骨细胞分化的机制研究

金晓丽，许 嘉，陈煊威，陈 瑾，黄 慧，张 婷，任 军，许 健   

  1. 浙江中医药大学医学技术与信息工程学院，浙江 杭州 310053
  • 出版日期:2023-11-20 发布日期:2023-12-08

Oridonin suppresses the effect of thioacetamide for promoting osteoclast differentiation of RAW264.7 cells and inhibiting osteoblast differentiation of bone mesenchymal stem cells

JIN Xiaoli, XU Jia, CHEN Xuanwei, CHEN Jin, HUANG Hui, ZHANG Ting, REN Jun, XU Jian   

  1. School of Medical Technology and Information Engineering, Zhejiang Chinese Medical University, Hangzhou 310053, China
  • Online:2023-11-20 Published:2023-12-08

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摘要： 目的 探究冬凌草甲素（ORI）和硫代乙酰胺（TAA）对破骨分化和成骨分化的作用及其机制。方法 CCK-8检测TAA和ORI对RAW264.7和骨髓间充质干细胞（BMSCs）增殖活力的影响；TRAP染色和免疫荧光染色检测TAA和ORI对RAW264.7破骨分化的影响；Western blot检测TAA和ORI对破骨特异性蛋白表达的影响；免疫荧光和流式细胞术检测TAA和ORI对p65核易位和活性氧生成的影响；碱性磷酸酶和茜素红染色检测TAA和ORI对BMSCs成骨分化的影响；Western blot法检测TAA和ORI对BMSCs成骨和凋亡相关蛋白表达的影响。结果 破骨诱导分化实验中，与TAA组相比，TAA+ORI组破骨细胞生成减少（P<0.01），MAPK/NF-κB通路明显被抑制（P<0.01），p65核易位水平显著降低，细胞内活性氧生成减少（P<0.01）。此外，与对照组相比，TAA能够抑制BMSCs ALP活性和钙化结节形成（P<0.01），并且诱导BMSCs凋亡。与TAA组相比，TAA+ORI组BMP-2/RUNX2表达增加，ALP活性增强（P<0.01），钙盐结节形成增加（P<0.01），细胞凋亡水平降低。结论 ORI可以调控MAPK/NF-κB预防TAA诱导的破骨分化，并且调控BMP-2/RUNX2缓解TAA抑制的骨形成。

关键词: 冬凌草甲素；硫代乙酰胺；破骨分化；成骨分化；骨质疏松

Abstract: Objective To explore the effect of oridonin (ORI) for suppressing thioacetamide (TAA)-induced osteoclast differentiation of RAW264.7 cells and antagonizing the inhibitory effect of TAA on osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Methods The effects of TAA and ORI on the proliferation of RAW264.7 cells and SD rat BMSCs were examined using CCK-8 assay. TRAP staining and immunofluorescence staining were used to observe the effects of TAA and ORI on osteoclast differentiation in RAW264.7 cells. The expressions of osteoclast-specific proteins in the treated cells were detected using Western blotting, and p65 nuclear translocation and reactive oxygen species (ROS) production in the cells were assessed with immunofluorescence assay and flow cytometry. Alkaline phosphatase (ALP) staining and alizarin red staining were used to examine the effects of TAA and ORI on osteogenic differentiation of BMSCs, and the expressions of osteogenic and apoptosis-related proteins in the cells were detected with Western blotting. Results Compared with RAW264.7 cells treated with TAA alone, the cells with the combined treatment with TAA and ORI showed decreased osteoclast differentiation (P<0.01) and significant inhibition of the MAPK/NF-κB pathway (P<0.01) with reduced p65 nuclear translocation and intracellular ROS production (P<0.01). In rat BMSCs, treatment with TAA alone significantly inhibited ALP activity and formation of calcified nodules (P<0.01) and induced obvious cell apoptosis. Compared with TAA-treated BMSCs, the cells treated with both TAA and ORI showed upregulated expressions of the BMP-2/RUNX2 pathway with enhanced ALP activity (P<0.01) and calcium deposition (P<0.01) and a lowered cell apoptosis level. Conclusion ORI inhibits TAA-induced osteoclast differentiation via regulating the MAPK/NF-κB pathway and antagonizes TAA-induced inhibition of bone formation by regulating the BMP-2/RUNX2 pathway

Key words: oridonin; thioacetamide; osteoclastogenesis; osteogenesis; osteoporosis