Cheng Xiaofei, Ni Bin, Zhang Feng, Hu Ying, Zhao Jie
Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China.
Department of Orthopedics, Changzheng Hospital, Second Military Medical University, Shanghai, China.
J Diabetes Res. 2016;2016:3765173. doi: 10.1155/2016/3765173. Epub 2016 Aug 22.
Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.
目的。研究高糖诱导的氧化应激是否与大鼠髓核细胞（NPCs）凋亡以及细胞外基质（ECM）代谢平衡相关关键基因的异常表达有关。方法。用不同浓度的葡萄糖培养NPCs以检测细胞活力和凋亡情况。用高糖（25 mM）培养的细胞不做处理或用N - 乙酰半胱氨酸或p38丝裂原活化蛋白激酶（MAPK）抑制剂SB 202190进行预处理。评估活性氧（ROS）的产生。通过蛋白质免疫印迹法检测p38 MAPK的激活情况。采用半定量逆转录聚合酶链反应（RT - PCR）分析ECM代谢相关基因的表达，这些基因包括Ⅱ型胶原、聚集蛋白聚糖、SRY相关高迁移率族蛋白盒9（Sox - 9）、基质金属蛋白酶3（MMP - 3）和金属蛋白酶组织抑制剂1（TIMP - 1）。结果。高糖降低了NPCs的活力并诱导凋亡。高糖导致ROS生成增加和p38 MAPK激活。此外，它对Ⅱ型胶原、聚集蛋白聚糖、Sox - 9和TIMP - 1的表达起负调节作用，对MMP - 3的表达起正调节作用。用N - 乙酰半胱氨酸或SB 202190预处理可改变这些结果。结论。高糖可能通过p38 MAPK依赖的氧化应激机制促进NPCs凋亡，触发ECM分解代谢途径并抑制其合成代谢活动。
