二烯丙基三硫诱导人髓样白血病HL-60细胞活性氧产生及其细胞毒性作用

Production and cytotoxicity of the reactive oxygen species induced by diallyl trisulfide in human myeloid leukemia HL-60 cells

目的:探讨二烯丙基三硫(DATS)诱导人白血病HL-60细胞产生活性氧(ROS)及其细胞毒性作用。方法:用浓度为50、100、150、200μmol/L DATS处理HL-60细胞1、3、6、12、24h后,流式细胞计数法检测HL-60细胞内的ROS水平,NBT还原实验分析NADPH氧化酶的活性,分别用NADPH氧化酶特异性阻断剂apocynin与抗氧化剂NAC(N-acetyl-L-cystein)预处理30min后,观察DATS对HL-60细胞产生活性氧的影响,分光光度法检测细胞质膜氧化产物丙二醛(MDA)与细胞蛋白氧化产物羰基化蛋白(protein carbonyl)。结果:DATS能诱导人白血病HL-60细胞产生ROS,当DATS处理细胞1-3h时,HL-60细胞产生ROS随DATS浓度的升高和处理时间的延长而升高,当浓度为150μmol/L DATS处理细胞3h时,ROS的荧光强度达到最高峰,其后维持在一个较高水平。NADPH氧化酶活性与ROS的产生一致。HL-60细胞的脂质过氧化和羰基化蛋白浓度与DATS诱导ROS产生的趋势基本一致,都在3h、150μmol/L处理点达到最高值。应用NADPH氧化酶特异性阻断剂apocynin或抗氧化剂NAC后,能显著降低HL-60细胞ROS的产生和细胞损伤。结论:NADPH氧化酶是DATS诱导HL-60细胞产生ROS的主要酶系,ROS能氧化细胞质膜和蛋白质。

AIM： To explore the production and cytotoxicity of the reactive oxygen species （ROS） induced by diallyl trisulfide （DATS） in HL -60 cells. METHODS： HL -60 cells were either treated with various doses of DATS alone, or DATS combination with apocynin, a specific NADPH oxidase inhibitor, or with antioxidant N - acetyl - L - cyste- ine （NAC） for 0, 1, 3, 6, 12 and 24 h, respectively. The intracellular ROS level was measured by flow cytometry. The activity of NADPH oxidase was evaluated by NBT reduction experiment. The content of both malondialdehyde （MDA） and the protein carbonyl were analyzed by spectrophotometer. RESULTS： The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL - 60 cells （ P 〈 0.05 ）, which was dose - and time - dependent. The fluorescence intensities of ROS reached at maximum when HL- 60 cells were incubated with 150 μmol/L DATS for 3 h. The NBT reduction experiment showed that DATS activated NADPH oxidase, the highest activity was observed when the cells were exposed to 150 μmol/L DATS for 3 h. DATS induced MDA and protein carbonyl production in HL -60 cells. Furthermore, both MDA and protein carbonyl reached the highest level in the cells exposed to 150μmol/L DATS for 3 h. Apocynin and NAC attenuated the production of MDA and protein carbonyl, suggesting that ROS induced by DATS was involved in the toxicity to the cells. CONCLUSION： DATS induces ROS production through activating NADPH oxidase in HL -60 cells. ROS increases the oxidation of membrane lipid and protein in HL -60 cells.