仙茅苔黑酚龙胆二糖苷调控NF-κB/Nrf2通路抑制破骨细胞氧化应激及其骨吸收的研究

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Factin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...

Orcinol gentiobioside from Curculigo orchioides inhibits oxidative stress and bone resorption in osteoclasts by regulation of NF-κB and Nrf2 pathways

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.