晚期糖基化终产物诱导ECV304细胞株氧化增强的可能机制

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。

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

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 O2-· was measured with cytochrome C,the level of reduced glutathione was measured with ThioGlo-1. Results O2-· increased from 1.37±0.67 nmol/(107·h) to 3.44±0.40,10.67±0.67 and 10.93±0.67 nmol/(107·h),and reduced glutathione decreased from 9.54±0.41 nmol/106 to 9.02±0.21,8.41±0.34,and 8.02±0.18 nmol/106 after 12.5 mg/L,50 mg/L,200 mg/L AGE-HSA stimulating;Apocynin,GF109203 and Genistein could inhibit these effects. Conclusion AGE-HSA could activate NADPH oxidatiate enzyme via protein kinase(PKC) and tyrosine protein kinase(TPK)to induce O2-· increasing and reduced glutathione decreasing and enhance intracellular oxidative effects.