晚期糖基化终产物诱导ECV304细胞株氧化增强的可能机制

Mechanism of Advanced Glycation End Products Induced Oxidative Effects in Cultured ECV304 Cells

目的探讨晚期糖基化终产物诱导细胞株ECV304氧化增强的机制。方法取ECV304细胞培养,与不同浓度的晚期糖基化终产物-人血清白蛋白共孵育,或分别经NADPH氧化酶抑制剂Apocynin或蛋白激酶C抑制剂GF109203或酪氨酸蛋白激酶抑制剂Genistein预孵育0.5 h后,再与晚期糖基化终产物—人血清白蛋白共孵育,1h后收集细胞,用细胞色素C法检测O2-.,ThioGlo-1试剂检测还原型谷胱甘肽。结果12.5 mg/L、50 mg/L和200mg/L晚期糖基化终产物—人血清白蛋白可导致ECV304细胞内O2-.从1.37±0.67 nmol/(107.h)增加到3.44±0.40、10.67±0.67和10.93±0.67 nmol/(107.h),使还原型谷胱甘肽从9.54±0.41 nmol/106降低到9.02±0.21、8.41±0.34和8.02±0.18 nmol/106,两者均呈剂量依赖性。Apocynin、GF109203及Genistein均可抑制O2-.的增加及还原型谷胱甘肽的降低。结论晚期糖基化终产物-人血清白蛋白可通过蛋白激酶C和酪氨酸蛋白激酶途径激活NADPH氧化酶,引起ECV304细胞内O2-.产生及还原型谷胱甘肽降低,导致细胞内氧化增强。

Aim To elucidate the mechanism of advanced glycation end products （AGE） inducing oxidative effects in cultured ECV304 cells. Methods ECV304 cells were cultured in vitro with AGE-human serum albumin（ HSA）, or pretreated with Apocynin or GF109203 or Genistein for 0.5 h, then cultured with AGE-HSA. After 1 h, the level of O^2-· was measuerd with cytochrome C, the level of reduced glutathione was measured with ThioGlo- 1. Results O^2- · increased from 1.37 ± 0. 67 nmol/（ 10^7. h） to 3.44 v 0.40, 10.67 ± 0.67 and 10.93 ±0.67 nmol/（ 10^7. h）, and reduced glutathione decreased from 9.54 ± 0.41 nmol/10^6 to 9.02 ± 0.21, 8.41 ± 0.34, mad 8.02± 0.18 nmol/10^6 after 12.5 mg/L, 50 mg/L, 200 mg/L AGE-HSA stimdating; Apocyrtin, GF109203 and Gertistein could inhibit these effects. Conchtsion AGE-HSA could activate NADPH oxidatiate enzyme via protein kinase （PKC） and tyrosine protein kinase （TPK）to induce O^2- · increasing and reduced glutathione decreasing and enhance inttracellular oxidative effects.