目的 探讨糖基化终产物(AGE)对肾间质成纤维细胞DNA损伤的影响及抗氧化剂的干预作用&#65377; 方法 不同浓度AGE修饰的牛血清白蛋白(AGE-BSA)作用于NRK-49F细胞24 h,普通培养基和牛血清白蛋白 (BSA)作为对照&#65377;N-乙酰半胱氨酸(NAC)预处理细胞以观察抗氧化剂的干预作用&#65377;AlamarBlue还原法测定细胞增殖活力,单细胞凝胶电泳(彗星实验)测定细胞DNA损伤&#65377; 结果 与对照组比较,AGE作用后的细胞增殖活力下降;DNA迁移距离(彗星尾长)增加,差异有统计学意义,且2者呈剂量依赖关系&#65377;各浓度BSA作用后对细胞增殖活力无明显影响;彗星尾长无明显变化&#65377;与相同浓度BSA相比, 400&#65380;800 mg/L AGE分别使AlamarBlue还原率下降14%和15%(P < 0.05);200&#65380;400&#65380;800 mg/L AGE组彗星尾长分别为BSA组的1.45&#65380;2.12&#65380;2.71倍(P < 0.05)&#65377;与未用NAC预处理组相比,NAC预处理可使AlamarBlue还原率上升[(45.15±0.93)% 比(38.40±0.81)%,P < 0.05];彗星尾长变短[(10.02±4.54) μm比(13.48±5.32) μm,P < 0.05]&#65377; 结论 AGE可导致肾间质成纤维细胞DNA损伤,使用抗氧化剂可有效减轻AGE导致的DNA损伤&#65377;细胞内氧化应激增强可能是AGE引起肾间质成纤维细胞DNA损伤的作用机制之一。

Objective To investigate the effects of advanced glycosylation end products (AGE) on DNA damage in cultured normal rat kidney fibroblasts (NRK-49F) cells, and the potential role of oxidative stress in AGEs-induced genotoxicity. Methods NRK-49F cells were treated with DMEM medium containing AGE-BSA (AGE-bovine serum albumin) at various concentrations (100, 200, 400, 800 mg/L) for 24 h. Cells were treated with serum-free DMEM medium or BSA at various concentration (100, 200, 400, 800 mg/L) for 24 h as control. To evaluate the potential role of oxidative stress in AGE-induced DNA damage, cells were preincubated with or without 10 mmol/L N-acetyl-l-cysteine (NAC) for 24 h and then were treated with AGE (400 mg/L) for another 24 h. Cell proliferation was measured by reduction of AlamarBlue. Single-cell gel electrophoresis (comet assay), a well-established method for quantifying DNA damage, was employed to analyze AGE-induced DNA damage. Results Compared with control medium and BSA, treatment with AGE caused significant inhibition of cell proliferation (P < 0.05)and increase of DNA damage (formation of comet) in a dose-dependent manner. Tail length of comet-formation caused by 200 mg/L AGE (P < 0.05) and 400 mg/L, 800 mg/L AGE (P < 0.01) was significantly longer than those of control group and BSA-treated group. Preincubation with antioxidant NAC attenuated AGE-induced DNA damage. Tail length of NAC pretreated group was significantly shorter than that of non-pretreated group (P < 0.05). However, treatment with BSA for 24 h did not show any increase in tail length (P > 0.05) and had no effect on cell proliferation (P > 0.05), compared with control medium. Conclusions AGE can induce DNA damage and inhibit cell proliferation in NRK-49F cells. Antioxidant can attenuate these effects. The enhanced oxidative stress may be one mechanism of underlying AGE-induced genotoxicity in chronic renal failure.