Effect Of Advanced Glycation End Products On Apoptosis Of Human Corneal Epithelial Cell

Objective:Delayed wound healing in diabetic cornea result in significant morbidity, prolonged hospitalization, and enormous health care expenses. Advanced glycation end products (AGEs) is the final product produced in the non-enzymatic catalysis reaction in the condition of continuous glucose, and plays a critical role in the progression of chronic complications in diabetic mellitus. Thus, we investigated the effect of AGEs on THCEs apoptosis in vitro, and the relationship with ROS to study the influence and mechanism of AGEs in delayed corneal wound healing due to diabetes.Methods:tolerated human corneal epithelial cells(THCE) were cultured in vitro with AGE-BSA of the concentrations of50,100,200,400ug/ml for6,12,24,48hours. Cells were stained with annexin V-Fitc and propidium iodide(PI). Flow cytometry was used to calculate the annexin V Fitc positive cells (early stage apoptotic cells) and Annexin V Fitc/PI positive cells(late apoptotic cells). Western blot were used to detect the expression of proapoptotic protein Bcl-2and Bax. After antioxidant NAC(20μmol/L)were used for1hour or DPI (10μM) or apocrynin300μM) were used for1hour, THCE were cultured with AGE-BSA of the best concentration and time. Flow cytometry was used to calculate the ratio of apoptosis. After DPI or apocrynin were used, Western blot were used to detect the expression of proapoptotic protein Bax. Results:Flow cytometry showed that the apoptotic rates in THCE cultured with50,100or200ug/ml AGE-BSA for6,12,24or48hours were significantly higher than those in control group(P＜0.05). The apoptotic rates in200ug/ml group for24hours were significantly higher than that in100ug/ml group(P＜0.05). The apoptotic rate increased along with the increase of culture time and concentration of AGE-BSA. Western blot showed200ug/ml AGE-BSA significantly increase Bax expression and suppressed the expression of Bcl-2in a time-dependent manner with the maximal effect by24h(P＜0.05), compared with control treatment. Pretreatment with DPI, apocrynin or NAC significantly decreased the apoptotic rate compared with the only AGE-BSA group(P＜0.05). Pretreatment with DPI or apocrynin dramatically inhibited AGE-induced expression of Bax (all P＜0.05).Conclusion:AGE-BSA may promote the apoptosis of THCE by the way of ROS which is generated by NADPH oxidase. AGE modification-induced pathobiological cascade may be involved in delayed corneal wound healing due to diabetes.