利用四唑盐染料WST反映巨噬细胞受脂多糖刺激活化后的氧化应激和代谢变化

摘要

目的通过四唑盐染料显色方法分析巨噬细胞受脂多糖刺激后的氧化应激和代谢变化，分析这种方法在炎症研究中的应用价值。　　方法通过活细胞计数方法和 CCK-8检测试剂分别检测RAW264.7巨噬细胞的活力；用 CCK-8和 WST-1显色方法研究超氧阴离子的释放；用 DCFH-DA 荧光染色和流式细胞术检测细胞内活性氧的生成；用乳酸检测试剂盒和烟酰胺腺嘌呤二核苷酸 NAD+(H)检测试剂盒分别检测上清中乳酸和细胞内 NADH 的含量。　　结果经脂多糖刺激的 RAW264.7细胞的活细胞计数结果和 CCK-8显色结果存在反差；相同数目的正常培养细胞和脂多糖刺激细胞的 CCK-8显色比较，后者读数显著升高，超氧化物歧化酶能够下调 CCK-8显色读数，WST-1显色结果与 CCK-8一致，脂多糖刺激细胞中的活性氧含量显著增加；脂多糖导致 RAW264.7细胞上清中的乳酸含量和细胞内的 NADH 含量显著增加。　　结论应用 WST 染料测定经脂多糖刺激的 RAW264.7细胞活力时，测定结果会受到细胞中还原性物质——超氧阴离子和 NADH 含量变化的干扰。通过 WST-1或 WST-8显色实验，可验证脂多糖能够刺激 RAW264.7巨噬细胞发生氧化应激和能量代谢变化。这一显色实验在炎症研究和药物筛选中可能有重要应用价值。%Objective To investigate the oxidative stress and metabolic changes in LPS-stimulated macrophages via the spectrophotometric assay based on WST tetrazolium salts, and analyze the application value of WST in inflammatory research. Methods Viability of RAW264.7 macrophages were determined by trypan blue excluding test and CCK-8 test, respectively. Superoxide production was measured by CCK-8 or WST-1. Reactive oxygen species were measured by flow-cytometry with DCFH-DA. Lactic acid production and reduced form of nicotineamide adenine dinucleotide (NADH) concentration were also determined. Results Inconsistent results of the LPS-stimulated RAW264.7 macrophages viability were obtained with trypan blue excluding test and CCK-8 assay. CCK-8 absorbance was significantly elevated in LPS-stimulated macrophages as compared to the same amount of macrophages cultured normally, which could be inhibited by superoxide dismutase (SOD). The result of WST-1 reduction was similar with CCK-8. Lactic acid in the culture medium, intracellular reactive oxygen species and NADH were all significantly increased by LPS. Conclusion When being used to test the LPS-stimulated macrophages, WST tetrazolium salt reduction reflecting cell viability was significantly disturbed by the content of reduced substances including the superoxide and NADH. WST tetrazolium salt reduction test might be a useful tool in the inflammation research and the anti-inflammation drug screening.