Tsao Chiung-Wen, Cheng Juei-Tang, Lin Yee-Shin
Department of Nursing, Chung Hwa College of Medical Technology, Tainan Hsien, Tainan, Taiwan, ROC.
Neuroimmunomodulation. 2002;10(6):328-36. doi: 10.1159/000071473.
Our previous work showed that 6-hydroxydopamine (6-OHDA) induced mouse thymocytes to undergo apoptosis both in vivo and in vitro. In the present study, we further investigated the mechanisms of 6-OHDA-induced apoptosis in vitro.
Naive mouse thymocytes were cultured with 6-OHDA. The percentages of apoptotic cells were quantified by propidium iodide staining, and DNA fragmentation was detected by agarose gel electrophoresis. Intracellular Bcl-2 was analyzed by immunofluorescence staining. Cu/Zn superoxide dismutase (Cu/Zn-SOD) activities were measured by the SOD-525 method.
The apoptotic effect of 6-OHDA was blocked by desipramine, a catecholamine uptake blocker. Treatment with 6-OHDA caused a reduction in Bcl-2 expression. VAD-FMK, a broad-spectrum caspase inhibitor, and DEVD-CHO, a potent inhibitor of caspase-3, could block 6-OHDA-induced thymocyte apoptosis. However, the specific caspase-1 (ICE) inhibitor YVAD-CMK had no effect. This cell death process was prevented by the protein synthesis inhibitor cycloheximide and by antioxidants. The level of Cu/Zn-SOD activities also decreased after cells were exposed to 6-OHDA.
These results suggest an apoptotic effect of 6-OHDA via the uptake of this neurotoxin by thymocytes, and that down-regulation of Bcl-2, activation of caspases, such as caspase-3 but not caspase-1, generation of reactive oxygen species, and new synthesis of proteins are involved in this process.
我们之前的研究表明，6-羟基多巴胺（6-OHDA）在体内和体外均可诱导小鼠胸腺细胞发生凋亡。在本研究中，我们进一步探讨了6-OHDA体外诱导凋亡的机制。
将未成熟的小鼠胸腺细胞与6-OHDA一起培养。通过碘化丙啶染色对凋亡细胞的百分比进行定量，并通过琼脂糖凝胶电泳检测DNA片段化。通过免疫荧光染色分析细胞内Bcl-2。采用SOD-525法测定铜/锌超氧化物歧化酶（Cu/Zn-SOD）活性。
去甲丙咪嗪（一种儿茶酚胺摄取阻滞剂）可阻断6-OHDA的凋亡作用。用6-OHDA处理导致Bcl-2表达降低。广谱半胱天冬酶抑制剂VAD-FMK和强效半胱天冬酶-3抑制剂DEVD-CHO可阻断6-OHDA诱导的胸腺细胞凋亡。然而，特异性半胱天冬酶-1（ICE）抑制剂YVAD-CMK无效。蛋白质合成抑制剂环己酰亚胺和抗氧化剂可阻止这种细胞死亡过程。细胞暴露于6-OHDA后，Cu/Zn-SOD活性水平也降低。
这些结果表明，6-OHDA通过胸腺细胞摄取这种神经毒素产生凋亡作用，并且Bcl-2的下调、半胱天冬酶（如半胱天冬酶-3而非半胱天冬酶-1）的激活、活性氧的产生以及蛋白质的新合成均参与了这一过程。
