Sakata N, Miyamoto K, Meng J, Tachikawa Y, Imanaga Y, Takebayashi S, Furukawa T
Second Department of Pathology, School of Medicine, Fukuoka University, Japan.
Atherosclerosis. 1998 Feb;136(2):263-74. doi: 10.1016/s0021-9150(97)00219-0.
To clarify the mechanism of cellular injury through the nonenzymatic reaction of glucose with proteins, we studied the cytotoxic effect of glycated bovine serum albumin on cultured smooth muscle cells in the presence of cupric ion. Glycated proteins were prepared by incubating bovine serum albumin with 0.5 M D-glucose in 0.3 M sodium phosphate buffer at 37 degrees C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-16, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreactivity of glycated proteins to anti-AGE antibody was 12-fold higher in g-BSA-16 than in g-BSA-2. Both g-BSA-2 and g-BSA-16 showed a concentration-dependent cytotoxicity in smooth muscle cells in the presence of 80 microM cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye reduction assay and dye exclusion test. Flow cytometry and spectrofluorophotometry using dihydrorhodamine 123 showed that the extracellular generation of oxidants was dose-dependently enhanced with increasing concentrations of g-BSA-2 or g-BSA-16 in the presence of cupric ion. However, no difference was observed in the intracellular generation of oxidants between the presence and absence of glycated proteins by flow cytometry using 2', 7'-dichlorofluorescein diacetate. Cytotoxicity and oxidant generation were prevented by catalase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells may be damaged by reactive oxygen species which are produced extracellularly by the interaction with the early glycation products and cupric ion, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth muscle cells.
为阐明葡萄糖与蛋白质的非酶促反应导致细胞损伤的机制，我们研究了糖化牛血清白蛋白在铜离子存在下对培养的平滑肌细胞的细胞毒性作用。通过将牛血清白蛋白与0.5M D-葡萄糖在0.3M磷酸钠缓冲液中于37℃孵育2、4和16周（分别为g-BSA-2、g-BSA-4和g-BSA-16）来制备糖化蛋白。孵育两周后形成了早期糖化产物，如果糖胺。然而，糖化蛋白对抗晚期糖基化终末产物抗体的免疫反应性在g-BSA-16中比在g-BSA-2中高12倍。通过MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑）染料还原试验和染料排斥试验，g-BSA-2和g-BSA-16在80μM铜离子存在下对平滑肌细胞均表现出浓度依赖性细胞毒性。使用二氢罗丹明123的流式细胞术和荧光分光光度法表明，在铜离子存在下，随着g-BSA-2或g-BSA-16浓度的增加，细胞外氧化剂的产生呈剂量依赖性增强。然而，使用二乙酸2',7'-二氯荧光素的流式细胞术未观察到糖化蛋白存在与不存在时细胞内氧化剂产生的差异。过氧化氢酶和钛铁试剂可预防细胞毒性和氧化剂的产生，但超氧化物歧化酶或羟基自由基清除剂甘露醇则不能。这些结果表明，平滑肌细胞可能被早期糖化产物与铜离子相互作用在细胞外产生的活性氧所损伤，并提示过氧化氢可能是导致平滑肌细胞这种氧化损伤的活性氧候选物。
