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  

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

作　　者：李鹤鸣 刘梦琴 虞艳玮 沈燚 杜金蔓 胡思婧 徐金龙 张泉龙 秦路平 张巧艳 LI Heming;LIU Mengqin;YU Yanwei;SHEN Yi;DU Jinman;HU Sijing;XU Jinlong;ZHANGQuanlong;QIN Luping;ZHANG Qiaoyan(School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;College of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350108,China;969 Hospital of PLA Joint Logistics Support Forces,Hohhot 010051,China)

机构地区：[1]浙江中医药大学药学院,浙江杭州310053 [2]福建中医药大学药学院,福建福州350108 [3]中国人民解放军联勤保障部队第九六九医院,内蒙古自治区呼和浩特010051

出　　处：《中草药》2024年第3期822-831,共10页Chinese Traditional and Herbal Drugs

基　　金：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)。

摘　　要：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H_(2)O_(2)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 H_(2)O_(2).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.

关 键 词：仙茅 苔黑酚龙胆二糖苷 破骨细胞 氧化应激 核因子-κB通路 核因子E2相关因子2通路 

分 类 号：R285.5[医药卫生—中药学]