Gao Z, Xu H G, Zhang X L, Wang H, Ma M M, Xiao L, Liu X
Department of Orthopedic Surgery, Yijishan Hosptial, Wannan Medical College, Wuhu 241001, China.
Zhonghua Yi Xue Za Zhi. 2016 Jul 19;96(27):2182-6. doi: 10.3760/cma.j.issn.0376-2491.2016.27.016.
To explore the relationship between nuclear factor (NF)-κB and vitro nature degeneration model of endplate chondrocyte in rats.
Rats endplate chondrocytes were isolated and cultured in vitro.Rebulated vitro natural degeneration model and cells were divided into control group (P2 cells group), naturally passaged group (P5 cells group) and NF-κB signaling pathway inhibition group (added Bay11-7082 when the passaged to P5 cells). The changes of cellular morphology were observed by inverted phase contrast microscope, the phenotype of endplate chondrocyte were identified by toluidine blue staining, electromagnetic compatibility (EMC) were observed by alcian blue staining.Type Ⅱ collagen, sry related HMG box (SOX)-9, matrix metalloproteinase(MMP)13 and aggrecan genes were detected by real time-polymerase chain reaction (RT-PCR) to verify the degeneration mode.NF-κB transcriptional activity was assessed by examining cytosolic phosphorylated IκBα and nuclear phosphorylated p65 levels by Western blot.
With chondrocytes passing, the cells lost the original morphology gradually.Alcian blue stains observed EMC decreased in naturally passaged group.The leave of Type Ⅱ collagen (P5/P2=0.182, P<0.01), aggrecan (P5/P2=0.287, P<0.01) and SOX-9 (P5/P2=0.488, P<0.01) were significantly reduced, MMP13 (P5/P2=1.324, P<0.05) significantly increased.Western blot analysis showed that in P5 cells nuclear phosphorylation p65 and cytosolic levels of phosphorylated IκBα increased and Bay11-7082 treatment attenuated increase in nuclear phosphorylation p65 and blocked acidinduced increase in cytosolic levels of phosphorylated IκBα.Moreover, the leave of Type Ⅱ collagen (Bay11-7082/P5=4.173, P<0.01), aggrecan (Bay11-7082/P5=2.732, P<0.05) and SOX-9 (Bay11-7082/P5=1.567, P<0.05) significantlyincreased, MMP13 (Bay11-7082/P5=0.611, P<0.05) significantly reduced in treatment group.
Inhibitor NF-κB signaling pathway plays an important role in the vitro degeneration of endplate cartilage.Reasonable regulation of NF-κB signaling pathway may be a new way to prevent Intervertebral disc degeneration.
探讨核因子（NF）-κB与大鼠终板软骨细胞体外自然退变模型的关系。
分离大鼠终板软骨细胞并进行体外培养。建立体外自然退变模型，将细胞分为对照组（P2细胞组）、自然传代组（P5细胞组）和NF-κB信号通路抑制组（传代至P5细胞时加入Bay11-7082）。通过倒置相差显微镜观察细胞形态变化，用甲苯胺蓝染色鉴定终板软骨细胞表型，用阿尔辛蓝染色观察细胞外基质（EMC）。采用实时聚合酶链反应（RT-PCR）检测Ⅱ型胶原蛋白、性别决定区Y框蛋白（SOX）-9、基质金属蛋白酶（MMP）13和聚集蛋白聚糖基因，以验证退变模型。通过蛋白质免疫印迹法检测细胞质中磷酸化IκBα和细胞核中磷酸化p65水平，评估NF-κB转录活性。
随着软骨细胞传代，细胞逐渐失去原有形态。自然传代组阿尔辛蓝染色观察到EMC减少。Ⅱ型胶原蛋白（P5/P2=0.182，P<0.01）、聚集蛋白聚糖（P5/P2=0.287，P<0.01）和SOX-9（P5/P2=0.488，P<0.01）的表达显著降低，MMP13（P5/P2=1.324，P<0.05）显著升高。蛋白质免疫印迹分析显示，P5细胞中细胞核磷酸化p65和细胞质中磷酸化IκBα水平升高，Bay11-7082处理减弱了细胞核磷酸化p65的升高，并阻断了酸诱导的细胞质中磷酸化IκBα水平的升高。此外，治疗组中Ⅱ型胶原蛋白（Bay11-7082/P5=4.173，P<0.01）、聚集蛋白聚糖（Bay11-7082/P5=2.732，P<0.05）和SOX-9（Bay11-7082/P5=1.567，P<0.05）的表达显著增加，MMP13（Bay11-7082/P5=0.611，P<0.05）显著降低。
抑制NF-κB信号通路在终板软骨体外退变中起重要作用。合理调控NF-κB信号通路可能是预防椎间盘退变的新途径。
