Asada S, Fukuda K, Nishisaka F, Matsukawa M, Hamanisi C
Department of Orthopaedic Surgery, Kinki University School of Medicine, Osaka-sayama, Osaka, Japan.
Inflamm Res. 2001 Jan;50(1):19-23. doi: 10.1007/s000110050719.
Recent observations demonstrated that reactive oxygen species facilitate cartilage degradation. We demonstrated that hydrogen peroxide (H2O2) caused inhibition of proteoglycan synthesis, induction of apoptosis and stimulation of extracellular signal-regulated protein kinase (ERK) of the chondrocytes (Inflamm Res 48: 399-403, 1999). To determine whether activation of ERK is involved in the induction of chondrocyte apoptosis, we examined the signal transduction pathways in this hydrogen peroxide induced apoptosis.
Bovine articular chondrocytes were cultured. To determine the induction of apoptosis, Annexin V staining and terminal deoxynucleotidyl transferase were used. The activity of caspase-3 was measured using an apopain assay kit. Intracellular Ca2+ imaging was observed after fura2-AM loading.
Hydrogen peroxide enhanced annexin V positive apoptotic cells and caspase-3 activity, which is an executor of apoptosis. Hydrogen peroxide also enhanced intracellular Ca2+ and preincubation with the intracellular Ca2+ chelator protected chondrocytes against hydrogen peroxide-induced cell apoptosis, indicating that an increase in the cytosolic Ca2+ plays a decisive role in this action. When ERK activity was blocked with geldanamycin and PD098059, increased apoptosis was evident.
Hydrogen peroxide induces chondrocyte apoptosis via Ca2+ signaling, and ERK is involved in these signal transduction pathways.
最近的观察表明活性氧促进软骨降解。我们证明过氧化氢（H2O2）可抑制蛋白聚糖合成、诱导细胞凋亡并刺激软骨细胞的细胞外信号调节蛋白激酶（ERK）（《炎症研究》48: 399 - 403, 1999）。为确定ERK的激活是否参与软骨细胞凋亡的诱导，我们研究了过氧化氢诱导的这种细胞凋亡中的信号转导途径。
培养牛关节软骨细胞。为确定细胞凋亡的诱导情况，使用膜联蛋白V染色和末端脱氧核苷酸转移酶。使用凋亡蛋白酶检测试剂盒测量caspase - 3的活性。在加载fura2 - AM后观察细胞内Ca2+成像。
过氧化氢增强了膜联蛋白V阳性凋亡细胞和caspase - 3活性，而caspase - 3是细胞凋亡的执行者。过氧化氢还增强了细胞内Ca2+，并且用细胞内Ca2+螯合剂预孵育可保护软骨细胞免受过氧化氢诱导的细胞凋亡，这表明胞质Ca2+的增加在这一过程中起决定性作用。当用格尔德霉素和PD098059阻断ERK活性时，细胞凋亡明显增加。
过氧化氢通过Ca2+信号诱导软骨细胞凋亡，并且ERK参与这些信号转导途径。
