周期性张应变通过活性氧生成促进膝关节骨关节炎患者的软骨细胞发生凋亡

Cyclic stretch-induced apoptosis in human osteoarthritic chondrocytes is mediated by reactive oxygen species generation

目的探讨周期性张应变诱导体外培养的膝关节骨关节炎(osteoarthritis,OA)患者软骨细胞发生凋亡过程中的活性氧(reactive oxygen species,ROS)的作用机制。方法对受力组软骨细胞施加频率0.5 Hz、强度20%延伸率的周期性张应变。对照组细胞的培养条件与受力组细胞一致,但不受力。N-乙酰半胱氨酸(NAC)和丁胱亚磺酰亚胺(BSO)干预组的软骨细胞在受力前分别使用NAC和BSO进行预处理。流式细胞术检测细胞凋亡率,DCFH-DA为荧光探针检测细胞内ROS含量,分光光度法检测软骨细胞内caspase-9活性变化。结果 0.5 Hz、20%延伸率的周期性张应变可以刺激软骨细胞内ROS、caspase-9活性及细胞凋亡率显著升高(P<0.05)。使用NAC和BSO抑制及升高细胞内ROS含量后,可以明显抑制及促进周期性张应变诱导的软骨细胞的caspase-9活性及细胞凋亡率(P<0.05)。结论周期性张应变通过促进ROS生成,进而激活caspase-9介导膝关节OA患者软骨细胞发生凋亡。抑制ROS的生成可以减轻周期性张应变导致的软骨细胞凋亡。

Objective To explore the mechanism of reactive oxygen species （ROS） generation on apoptosis of human osteoarthritic chondrocytes induced by cyclic stretch in vitro. Methods The human osteoarthritic chon- drocytes were subjected to cyclic stretch at the frequency of 0.5 Hz with 20% elongation. The chondrocytes with- out cyclic stretch were used as a control. ROS generation in chondrocytes was inhibited by the antioxidant, N-ac- etyI-L-cysteine （NAC） and potentiated by the glutathione depleter, DL-buthionine-[ S, R ]-sulfoximine （BSO）. Apoptosis was detected by flow cytometry. Intracellular ROS was detected using DCFH-DA and caspase-9 activi- ty was measured using spectrophotometry. Results The cyclic stretch at the frequency of 0.5 Hz with 20% elon- gation induced ROS generation, and activation of caspase-9 and apoptosis in human osteoarthritic chondrocytes were significantly increased （ P 〈 0.05）. The inhibition or potentiation of intracellular ROS by NAC or BSO could obviously inhibit or improve caspase-9 activity and apoptosis in chondrocytes under cyclic stretch （P 〈0.05）. Conclusions Cyclic stretch-induced apoptosis in human osteoarthritic chondrocytes is mediated by ROS genera-tion and activation of caspase-9. Suppression of ROS can prevent chondrocytes from apoptosis induced by cyclic stretch,