Wang S J, Sun Z Y, Liu C, Yan P, Liang H, Huang K, Wang D G, Li Y, Tian J W
Department of Orthopedics, Shanghai General Hospital Affiliated to Shanghai Jiaotong University, School of Medicine, Shanghai 201620, China.
Zhonghua Yi Xue Za Zhi. 2017 Jul 4;97(25):1964-1969. doi: 10.3760/cma.j.issn.0376-2491.2017.25.010.
To investigate the effect of high mechanical stretch stress(HMS)on human nucleus pulposus cells and its regulatory mechanism. The non-degenerated nucleus pulposus tissue (Pfirrmann<grade Ⅲ) removed from the patient's surgery was harvested and the human nucleus pulposus cells were isolated and cultured. In the presence or absence of pretreatment with the NF-κB specific blocker Bay11-7082, the cultured human nucleus pulposus cells were loaded cyclic mechanical stretch stress(CMS) with different parameters using the Flexercell system.The cell culture medium was collected and the secretion of inflammatory cytokines was detected by Elisa. The nucleus pulposus cells loaded with cyclic mechanical stretch stress(CMS) was collected, the changes of NF-κB/P65 signal pathway were detected, The mRNA and protein levels' expression changes were detected by RT-PCR and WB; after human nucleus pulposus cells were exposed to IL-1β, with or without Bay11-7082, the changes of P65 were detected by immunofluorescence. Those mechanical stretch stress of high amplitude (9%, 19%), low frequency (0.01 Hz) and long duration (72 h) led to degeneration of human nucleus pulposus cells, while the mechanical stretch stress of low amplitude (3%), low frequency and long duration could not promote the degeneration process; the mechanical stretch stress of high amplitude(19%), low-frequency(0.01 Hz) could promote the release of inflammatory cytokines of human nucleus pulposus cells after 24 h duration; high-amplitude, low-frequency mechanical stretch stress could activate the NF-κB signaling pathway in human nucleus pulposus cells, Bay11-7082 could block the process; immunofluorescence showed that IL-1β could promote the phosphorylation of P65 in the cytoplasm of human nucleus pulposus cells and promote the entry of P65 into the cell nucleus process, Bay11-7082 could block those processes; Bay11-7082, the specific blocking agent of NF-κB signaling pathway, could block the degeneration process of human nucleus pulposus cells induced by high cyclic mechanical stretch stress(CMS) in a dose-dependent. High cyclic mechanical stretch stress promotes human nucleus pulposus cells degeneration through NF-κB signaling pathway.
探讨高机械拉伸应力（HMS）对人髓核细胞的影响及其调控机制。收集患者手术中切除的未退变髓核组织（Pfirrmann分级<Ⅲ级），分离培养人髓核细胞。在存在或不存在NF-κB特异性阻断剂Bay11-7082预处理的情况下，使用Flexercell系统对培养的人髓核细胞施加不同参数的周期性机械拉伸应力（CMS）。收集细胞培养基，通过酶联免疫吸附测定法检测炎性细胞因子的分泌。收集施加周期性机械拉伸应力（CMS）的髓核细胞，检测NF-κB/P65信号通路的变化，通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（WB）检测mRNA和蛋白质水平的表达变化；人髓核细胞暴露于白细胞介素-1β（IL-1β）后，在有或没有Bay11-7082的情况下，通过免疫荧光检测P65的变化。那些高幅度（9%，19%）、低频率（0.01Hz）和长时间（72小时）的机械拉伸应力导致人髓核细胞退变，而低幅度（3%）、低频率和长时间的机械拉伸应力不能促进退变过程；高幅度（19%）、低频率（0.01Hz）的机械拉伸应力在持续24小时后可促进人髓核细胞炎性细胞因子的释放；高幅度、低频率的机械拉伸应力可激活人髓核细胞中的NF-κB信号通路，Bay11-7082可阻断该过程；免疫荧光显示IL-1β可促进人髓核细胞质中P65的磷酸化并促进P65进入细胞核的过程，Bay11-7082可阻断这些过程；NF-κB信号通路的特异性阻断剂Bay11-7082可剂量依赖性地阻断高周期性机械拉伸应力（CMS）诱导的人髓核细胞退变过程。高周期性机械拉伸应力通过NF-κB信号通路促进人髓核细胞退变。
