摘要
目的 基于Nrf2/HO-1通路探讨健骨颗粒含药血清对氧化应激状态成骨细胞骨形成的调控机制。方法 取6周龄SPF级雄性SD大鼠40只,随机分为健骨颗粒组和生理盐水组各20只。健骨颗粒组每天灌服含生药量7.8 g/kg健骨颗粒浸膏稀释液2 mL,生理盐水组每天灌服生理盐水2 mL,2组每日灌胃1次,灌胃7 d后取大鼠腹主动脉血制备健骨颗粒含药血清和生理盐水血清。将课题组前期选用UMR-106细胞构建的Nrf2敲减稳定株细胞和Nrf2阴性对照细胞,分别分为Nrf2-KD+JG组、Nrf2-KD+NS组和NC+JG组、NC+NS组。Nrf2-KD+JG组和NC+JG组分别采用10%健骨颗粒含药血清加高糖DMEM培养12 h;Nrf2-KD+NS组和NC+NS组分别采用10%生理盐水血清加高糖DMEM培养12 h,再用10μmol/L过氧化氢干预12 h;采用超氧化物阴离子荧光探针(DHE)检测4组超氧化物阴离子含量,茜素红染色实验观察4组矿化结节数量,Western blot检测4组Nrf2、核Nrf2、HO-1、RUNX2蛋白相对表达量。结果 与NC+JG组比较,Nrf2-KD+JG组超氧化物阴离子含量增多(P<0.05),显微镜下可见矿化结节数量减少,Nrf2、核Nrf2、HO-1和RUNX2蛋白相对表达量均明显降低(P<0.05)。与NC+NS组比较,Nrf2-KD+NS组超氧化物阴离子含量增多,显微镜下可见矿化结节数量减少,4个指标蛋白相对表达量均明显降低(P<0.05),NC+JG组超氧化物阴离子含量显著降低(P<0.05),显微镜下可见矿化结节数量增多,4个指标蛋白相对表达量均明显升高(P<0.05)。结论 健骨颗粒可以激活成骨细胞内Nrf2/HO-1信号通路,降低细胞内氧化应激水平,增强细胞矿化能力,促进成骨细胞骨形成。
Objective:The regulatory mechanism of serum containing Jiangu Granules on bone formation of osteoblasts under oxidative stress was discussed based on Nrf2/HO-1 pathway.Methods:A total of forty SPF male SD rats aged 6 weeks were randomly divided into Jiangu Granules group and normal saline group,with 20 rats in each group.Jiangu Granules group was given 2 mL of Jiangu Granules extract diluent containing 7.8 g/(kg·d)of crude drug,while saline group was given 2 mL of saline,by gavage once a day respectively.After 7 days of gavage,the abdominal aorta blood of rats was taken to prepare Jiangu Granules drug-containing serum and saline serum.The Nrf2 knock-down stable cells and Nrf2 negative control cells constructed by UMR-106 cells were divided into Nrf2-KD+JG group,Nrf2-KD+NS group,NC+JG group and NC+NS group respectively.The Nrf2-KD+JG and NC+JG groups were cultured with high glucose DMEM containing 10%serum of Jiangu Granules for 12 h,while the Nrf2-KD+NS and NC+NS groups were cultured with high glucose DMEM containing 10%saline serum for 12 h,and then were intervened with 10μmol/L hydrogen peroxide for 12 h.Dihydroethidium(DHE)fluorescent probe was used to detect the content of superoxide anion in four groups,alizarin red staining experiment was used to observe the number of mineralized nodules in four groups,and Western blot was used to detect the relative expression of Nrf2,nuclear Nrf2,HO-1 and RUNX2 proteins in four groups.Results:Compared with the NC+JG group,the content of superoxide anion in the Nrf2-KD+JG group increased(P<0.05),the number of mineralized nodules decreased under microscope,and the relative expressions of Nrf2,nuclear Nrf2,HO-1 and RUNX2 proteins decreased significantly(P<0.05).Compared with the NC+NS group,the content of oxide anions in the Nrf2-KD+NS group increased,the number of mineralized nodules decreased under microscope,and the relative expression of four index proteins decreased significantly(P<0.05).The content of superoxide anion in the NC+JG group decreased significantly(P<0.05),the number of mineralized nodules increased under microscope,and the relative expression of four index proteins increased significantly(P<0.05).Conclusion:Jiangu Granules can activate the Nrf2/HO-1 signaling pathway in osteoblasts,reduce the level of intracellular oxidative stress,enhance the mineralization ability of cells and promote the bone formation of osteoblasts.
作者
周芬
孙攀
黄云梅
王志强
林燕萍
ZHOU Fen;SUN Pan;HUANG Yunmei;WANG Zhiqiang;LIN Yanping(Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Fujian Key Laboratory of Integrative Medicine on Geriatrics,Fuzhou,Fujian 350122,China;Traditional Chinese Medicine Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200071,China)
出处
《福建中医药》
2024年第1期10-14,28,共6页
Fujian Journal of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(81574003)
福建省财政专项(X2019002)。
