力学刺激与淫羊藿苷耦合作用抑制疲劳损伤诱导破骨细胞分化的分子机制研究  

Molecular mechanism of the combination of mechanical strain stimulation and icariin on inhibiting the differentiation of osteoclasts induced by fatigue load stimulation

作　　者：刘立丽[1] 骆雄飞[1] 刘斯文[1] 赵娜[1] 张西正 王强松[3] Liu Lili;Luo Xiongfei;Liu Siwen;Zhao No;Zhang X izhe ng;Wang Qiangs ong(Department of Tuina, The First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;Institute of Medical Service and Technology, Academy of Military Science, Tianjin 300161, China;Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China)

机构地区：[1]天津中医药大学第一附属医院推拿科,300193 [2]军事科学院系统工程研究院卫勤保障技术研究所,天津300161 [3]中国医学科学院北京协和医学院生物医学工程研究所,天津300192

出　　处：《国际生物医学工程杂志》2018年第5期386-389,394,共5页International Journal of Biomedical Engineering

基　　金：国家自然科学基金(31500761,81674093,81503672);国家自然科学基金重点项目(11432016).

摘　　要：目的 探讨力学刺激与淫羊藿苷(ICA)耦合作用对疲劳载荷下破骨细胞分化的影响及可能存在的分子机制。方法 体外培养小鼠单核巨噬细胞系RAW264.7,空白对照组为α-MEM完全培养基;疲劳载荷下破骨细胞模型组更换为破骨细胞培养液(含质量浓度为40 ng/ml巨噬细胞集落刺激因子和40 ng/ml破骨细胞分化因子的α-MEM完全培养基),并对RAW264.7细胞施加5 000 με基底拉伸应变;力学刺激+ICA组经上述细胞因子和高应变(5 000 με)刺激后,更换为含有1×10^-5 mol/L ICA的α-MEM完全培养基,并施加1 000 με基底拉伸应变。采用抗酒石酸酸性磷酸酶(TRAP)检测试剂盒检测破骨细胞中TRAP的活性变化;实时定量PCR检测破骨细胞标志性基因TRAP、组织蛋白酶K(CTSK)和基质金属蛋白酶9(MMP-9)的mRNA表达;蛋白质印迹法(Western Blot)分析核因子κB(NF-κB)异位情况。结果 与模型组相比,力学刺激(1 000 με的基底拉伸)和ICA(1×10^-5 mol/L)耦合作用能显著抑制破骨细胞中TRAP的活性(P<0.01),减少破骨细胞的形成;显著下调标志性基因TRAP、CTSK和MMP-9的mRNA表达,差异均具有统计学意义(均P<0.01);通过抑制NF-κB信号通路中P65、P50、NF-κB抑制蛋白-α(IκB-α)的磷酸化来抑制破骨细胞的生成。结论 力学刺激与ICA耦合作用可有效抑制疲劳载荷下破骨细胞的分化及其骨吸收功能,其作用机制可能是通过调节NF-κB信号通路来实现。Objective To investigate the effect and molecular mechanism of the combination of mechanical strain stimulation and icariin (ICA) on inhibiting the differentiation of osteoclasts induced by fatigue load stimulation. Methods The mouse mononuclear macrophage cell line RAW264.7 was cultured in vitro, and the blank control group was α-MEM complete medium. In the fatigue load group, RAW264.7 cells were treated with 5 000 με mechanical stretch strain, and then cultured in an osteoclast culture medium that was an α-MEM complete medium containing 40 ng/ml macrophage colony-stimulating factor and 40 ng/ml osteoclast differentiation factor. In the mechanical stimulation + ICA group, RAW264.7 cells were treated as the same procedure in the fatigue load group, and then cultured in an α-MEM complete medium containing 1×10^-5 mol/L ICA simultaneously with a 1000 με tensile strain on the substrate. The activity of tartrate-resistant acid phosphatase (TRAP) was detected using a TRAP assay kit. The mRNA expression of the osteoclast marker genes, i.e. TRAP, cathepsin K(CTSK) and matrix metalloproteinase 9 (MMP-9) was detected by real-time RT-PCR. The nuclear translocation of nuclear factor kappa B (NF-κB) was analyzed by Western Blot. Results Compared with the fatigue load group, the combination of mechanical stimulation (1 000 με substrate stretching) and ICA (1×10^-5 mol/L) could significantly inhibit the activity of TRAP in osteoclasts (P<0.01) and reduce osteoclastosis. Moreover, that combination not only could down-regulate the mRNA expression of TRAP, CTSK and MMP-9 and the differences were statistically significant (all P<0.01), but also could inhibit the formation of osteoclasts by inhibiting the phosphorylation of P65, P50 and IκB-α in NF-κB signaling pathway. Conclusions The coupling of mechanical stimulation and ICA can effectively inhibit the osteoclast differentiation and the bone resorption induced by fatigue load, and the mechanism may involve regulating NF-κB signaling pathway.

关 键 词：力学刺激 淫羊藿苷 破骨细胞 核因子ΚB 

分 类 号：R318.01[医药卫生—生物医学工程]