Xu Hong-Guang, Ma Ming-Ming, Zheng Quan, Shen Xiang, Wang Hong, Zhang Shu-Feng, Xu Jia-Jia, Wang Chuan-Dong, Zhang Xiao-Ling
Department of Spine Surgery, Yijishan Hospital, Wannan Medical College, Wuhu, Anhui, China.
Department of Orthopedic Surgery, The People's Hospital of Luan, Luan, Anhui, China.
Spine (Phila Pa 1976). 2016 Aug 15;41(16):1261-1271. doi: 10.1097/BRS.0000000000001532.
The changes of endplate chondrocytes induced by intermittent cyclic mechanical tension (ICMT) were observed by realtime reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis.
To investigate the role of RhoA/ROCK-1 signaling pathway and E-cadherin/P120-catenin complex in endplate chondrocytes degeneration induced by ICMT.
ICMT can induce the endplate chondrocyte degeneration. However, the relationship between P120-catenin or RhoA/ROCK-1 signaling pathway and endplate chondrocytes degeneration induced by ICMT is not clear.
ICMT (strain at 0.5 Hz sinusoidal curve at 8% elongation) was applied to rat endplate chondrocytes for 6 days, 16 hours a day. The cell viability and apoptosis were examined by the LIVE/DEAD assay and flow cytometry. Histological staining was used to examine the lumbar disc tissue morphology and extracellular matrix. To regulate RhoA/ROCK-1 signaling pathway and the expression of E-cadherin and P120-catenin, RhoA/ROCK-1 pathway-specific inhibitors, E-cadherin, and p120-catenin plasmid were applied. Coimmunoprecipitation was employed to examine the interaction between E-cadherin and P120-catenin, P120-catenin, and RhoA. The related gene expression and protein location was examined by realtime reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence.
There was no change of viability verified by LIVE/DEAD assay and flow cytometry after ICMT loading. ICMT loading led to RhoA/ROCK-1 signaling activation and the loss of the chondrogenic phenotype of endplate chondrocytes. Inhibition of RhoA/ROCK-1 signaling pathway significantly ameliorated the degeneration induced by ICMT. The expression of P120-catenin and E-cadherin were inhibited by ICMT. ICMT reduced the interaction between P120-catenin and E-cadherin. Furthermore, over-expression of P120-catenin and E-cadherin can suppress the expression of chondrogenic gene, over-expression of P120-catenin can suppress the RhoA/ROCK-1 signaling pathway, but over-expression of E-cadherin cannot do it.
P120-catenin protects endplate chondrocytes from ICMT Induced degeneration by inhibiting the expression of RhoA/ROCK-1 signaling pathway.
N/A.
通过实时逆转录-聚合酶链反应、免疫荧光和蛋白质免疫印迹分析观察间歇性循环机械张力（ICMT）诱导的终板软骨细胞的变化。
探讨RhoA/ROCK-1信号通路和E-钙黏蛋白/P120-连环蛋白复合物在ICMT诱导的终板软骨细胞退变中的作用。
ICMT可诱导终板软骨细胞退变。然而，P120-连环蛋白或RhoA/ROCK-1信号通路与ICMT诱导的终板软骨细胞退变之间的关系尚不清楚。
将ICMT（0.5赫兹正弦曲线，8%伸长率的应变）施加于大鼠终板软骨细胞，每天16小时，持续6天。通过活/死细胞检测法和流式细胞术检测细胞活力和凋亡情况。采用组织学染色检查腰椎间盘组织形态和细胞外基质。为调节RhoA/ROCK-1信号通路以及E-钙黏蛋白和P120-连环蛋白的表达，应用了RhoA/ROCK-1通路特异性抑制剂、E-钙黏蛋白和p120-连环蛋白质粒。采用免疫共沉淀法检测E-钙黏蛋白与P120-连环蛋白、P120-连环蛋白与RhoA之间的相互作用。通过实时逆转录-聚合酶链反应、蛋白质免疫印迹和免疫荧光检测相关基因表达和蛋白质定位。
ICMT加载后，活/死细胞检测法和流式细胞术验证细胞活力无变化。ICMT加载导致RhoA/ROCK-1信号激活以及终板软骨细胞软骨形成表型丧失。抑制RhoA/ROCK-1信号通路可显著改善ICMT诱导的退变。ICMT抑制P120-连环蛋白和E-钙黏蛋白的表达。ICMT减少了P120-连环蛋白与E-钙黏蛋白之间的相互作用。此外，P120-连环蛋白和E-钙黏蛋白的过表达可抑制软骨形成基因的表达，P120-连环蛋白的过表达可抑制RhoA/ROCK-1信号通路，但E-钙黏蛋白的过表达则不能。
P120-连环蛋白通过抑制RhoA/ROCK-1信号通路的表达保护终板软骨细胞免受ICMT诱导的退变。
无。
