糖基化终产物对肾间质成纤维细胞DNA损伤的影响

Advanced glycosylation end products induce DNA damage in NRK-49F cells

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。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24h，普通培养基和牛血清白蛋白（BSA）作为对照。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。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。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。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。与相同浓度BSA相比，400、800ms／LAGE分别使AlamarBlue还原率下降14％和15％（P〈0．05）；200、400、800mg／LAGE组彗星尾长分别为BSA组的1．45、2．12、2．71倍（P〈0．05）。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]。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。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, ceils 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.