目的 探讨仙茅苔黑酚龙胆二糖苷（orcinol gentiobioside，OGB）对氧化应激诱导的破骨细胞（osteoclasts，OCs）的作用及机制。方法 可溶性核因子-κB受体活化因子配体（soluble receptor activator of nuclear factor-κB ligand，sRANKL）联合H₂O₂诱导RAW264.7细胞，建立氧化应激诱导的OCs模型；CCK-8法检测OCs活力；抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色测定OCs的数目；磷酸苯二钠法测定OCs的TRAP活性；免疫荧光法观察OCs的F-actin环结构和形态，以及p65和核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）的核转位；ELISA法测定OCs相关的生化指标；Western blotting检测骨吸收关键蛋白及核因子-κB（nuclear factor-κB，NF-κB）和Nrf2通路相关蛋白的表达。结果 OGB显著抑制氧化应激诱导的OCs的形成和分化（P＜0.01），并抑制TRAP活性、F-actin环的形成及其骨吸收作用（P＜0.05、0.01），下调骨吸收关键蛋白c-Fos、组织蛋白酶K（cathepsin K，CTSK）、基质金属蛋白酶9（matrix metalloprotein 9，MMP9）的表达（P＜0.05、0.01），降低活性氧（reactive oxygen species，ROS）和还原型辅酶II（nicotinamide adenine dinucleotide phosphate，NADPH）氧化酶4（NADPH oxidase 4，NOX4）的活性（P＜0.05、0.01），增加抗氧化酶血红素加氧酶-1（heme oxygenase-1，HO-1）、醌氧化还原酶1（quinone oxidoreductase 1，NQO1）、γ-谷氨酰半胱氨酸合成酶（gamma-glutamyl cysteine synthetase，γ-GCS）的活性（P＜0.05、0.01），增强OCs中Nrf2的表达和核转位，抑制肿瘤坏死因子受体相关蛋白6（tumor necrosis factor receptor-associated factor 6，TRAF6）的募集、核因子-κB抑制因子α（inhibitor of NF-κB-α，IκBα）的降解，并阻止p65的磷酸化和核转位（P＜0.05）。结论 OGB能够通过调控NF-κB和Nrf2通路，抑制氧化应激诱导的OCs形成分化和骨吸收作用。

Objective To investigate the effect and mechanism of orcinol gentiobioside (OGB) from Curculigo orchioides on osteoclasts (OCs) induced by oxidative stress. Methods OCs model were established from RAW264.7 cells induced by soluble receptor activator of nuclear factor-κB ligand (sRANKL) and H₂O₂. The viability of OCs was assayed by CCK-8 method. The number of OCs was determined by tartrate-resistant acid phosphatase (TRAP) staining. TRAP activity of OCs was detected by using p-PNPP-Na method. F-actin and nuclear translocation of p65 and nuclear factor erythroid 2-related factor 2 (Nrf2) were stained with immunofluorescence method. The biochemical parameters of OCs were detected by ELISA. The expressions of key proteins involved in bone resorption and nuclear factor-κB (NF-κB)/Nrf2 pathway were analyzed by Western blotting. Results OGB significantly inhibited the formation and differentiation of OCs (P < 0.01), and significantly suppressed TRAP activity, formation of F-actin and bone resorption of OCs (P < 0.05, 0.01), down-regulated the expressions of c-Fos, cathepsin K (CTSK) and matrix metallopeptidase 9 (MMP9) (P < 0.05, 0.01), reduced the levels of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) activity (P < 0.05, 0.01), improved the activities of heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO1) and gamma-glutamyl cysteine synthetase (γ-GCS) (P < 0.05, 0.01). In addition, OGB also enhanced the expression and nucleus translocation of Nrf2 in OCs, inhibited the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6), the degradation of inhibitor of NF-κB-α (IκBα) and prevented the phosphorylation and nuclear translocation of p65 in OCs (P < 0.05). Conclusion OGB inhibits formation, differentiation and bone resorption of OCs induced by oxidative stress through regulation of NF-κB and Nrf2 pathways.

国家自然科学基金资助项目（81973534）；国家自然科学基金资助项目（82374003）