背景：甲基乙二醛 (Methylglyoxal；MG) 為一致突變物，體內的糖質新生、葡萄糖的糖解作用和蛋白質的糖化作用中皆會產生，在高血糖症、發炎反應、尿毒症、老化及氧化壓力下，都可以觀察到血漿中的MG濃度明顯上升。MG在組織中的累積，會誘發DNA和蛋白質發生糖基化 (glycation)，進一步形成DNA－蛋白質交叉聯結(DNA－protein cross-link；DPC)，嚴重影響基因轉錄，甚至造成細胞凋亡，這些現象與糖尿病合併症的發展關係密切，但MG如何誘發細胞內DPC形成的分子機制仍不清楚。目的：本研究採用人類血管內皮類似細胞 (ECV304)，探討MG造成DPC形成的可能機制。方法：以彗星分析法測量細胞內DPC程度；以DCHF-DA染色法觀察細胞內活性氧物種 (reactive oxygen species；ROS)；以免疫圓點染色法測量蛋白質羰基化程度；以MTT分析法觀察細胞活性；以結晶紫染色法計算細胞總數，觀察細胞增生。結果：MG在ECV304細胞及無細胞 (cell-free) 系統，都循時間及劑量增加方式，明顯增加DPC的形成，但MG (0.5~4 mM) 0.5~3小時處理，卻不會使ECV304細胞內ROS明顯增加。在處理MG (2 mM) 2小時的同時加入抗氧化劑aminoguanidine (AG)、glutathione (GSH)、N-acetyl-L-cysteine (NAC)、ascorbic acid (VitC)、curcumin (Cur)或lipoic acid (LA)，發現具清除MG能力的抗氧化劑AG、GSH及NAC才能有效降低MG造成的DPC，反之不具清除MG能力的抗氧化劑VitC、Cur及LA則否。MG也會造成ECV304細胞內蛋白質羰基化隨劑量上升而增加，2 mM MG造成羰基化在2小時達到高峰，之後羰基化程度會快速降低。以蛋白分解體 (proteasome) 抑制劑 (ALLN) 前處理細胞24小時，會更增加MG在ECV304細胞所導致的DPC。不同MG濃度 (0.5~10 mM) 處理細胞2小時後，發現細胞活性的IC50約為9.4 mM。細胞處理MG 2小時後，再培養於新鮮培養基中48小時，發現細胞總數的IC50約為0.5 mM，此現象顯示MG短時間處理，並不會造成細胞的立即死亡，但卻明顯抑制細胞的分裂。在試管試驗中，MG可以直接羰基化小牛血清白蛋白，並使之與小牛胸腺DNA發生交叉聯結。結論：綜合上述結果， MG造成ECV304細胞產生DPC的原因，可能與ROS關係微弱，而是透過MG對DNA或蛋白質產生羰基化修飾，造成DPC產生。並且，DPC的形成對細胞的立即性傷害不大，卻會明顯降低細胞的增生。
Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced from glycolytic intermediates and gluconeogenesis. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG can modify both DNA and proteins and induces a crosslink between DNA and protein; however, its mechanism is still unclear. In this study, we investigated the possible mechanisms of MG-induced DNA-protein crosslink (DPC) in ECV304 human endothelial like cells. Cells were exposed to various concentrations (0.5-4 mM) of MG for 0.5-3 hours. DPC and protein carbonylation of MG-treated cells were assayed by modified comet assay and immuno-dot blot assay, respectively. The level of intracellular peroxide of MG-treated cells was also detected by DCHF-DA assay using a multi-well fluorescence plate reader. MG treatment caused a time- and dose-dependent increase of DPC in ECV304 cells. MG also caused a dose-dependent increase of protein carbonylation in ECV304 cells. An addition of 2 mM MG resulted in a transient increase in protein carbonylation in ECV304 cells, and this increase reached a peak within 2 hours and then rapidly decreases. Co-treatment carbonyl scavenging drugs, such as aminoguanidine, glutathione, and N-acetyl-L-cysteine and MG in ECV304 cells significantly inhibited the formation of DPC, whereas co-treatment with antioxidants ascorbic acid, curcumin, and lipoic acid did not decrease MG-induced DPC in ECV304 cells. Although MG induced significant increase in DPC and protein carbonylation levels, it did not change the ROS level significantly in ECV304 cells. Co-treatment proteasome inhibitor, such as ALLN, and NU1025 and MG in ECV304 cells increase the formation of DPC. Taken together, these results suggest that MG induces DPC formation in ECV304 cells via a ROS-independent protein carbonylation pathway.
