| BackgroundDiabetic cataract, as one of major diabetes complications, is among the most common blind-leading ophthalmopathy which is only secondary to diabetic retinopathy. Within human body of diabetes patients, the binding between large amounts of accumulated advanced glycation end-products (AGEs) and its receptor—receptor for advanced glycation end-products (RAGE) is able to increase the synthesis of reactive oxygen species (ROS) and enhance the transcription activity of nuclear factor, thus leading to oxidative damage of tissue and inflammatory reaction. That pathway may account for the important function of AGEs in various pathological conditions caused by diabetes. ObjectivesTo investigate the RAGE expression in lens epithelial cells (LECs) of diabetic cataract (DC), age-related cataract (ARC) and normal lenses, to observe the effect of AGEs on oxidative state and RAGE level in LECs. The possible role of AGEs-RAGE interaction in the pathogenesis of diabetic cataract was also discussed, which may provide more theoretical evidences for basic research and medical treatment of diabetic cataract. Methods(1) The anterior capsules of seventy lenses from diabetic cataract patients and seventy-two lenses from age-related cataract patients during cataract surgery and twenty-four lenses from donor eyes were collected and the expression of RAGE in those capsules was examined by immunohistochemistry. The expression rates of RAGE were analyzed in LECs of diabetic cataract, age-related cataract and normal lenses. Meanwhile, all diabetic cataract patients'datum including sex, age, diabetes duration, level of glycosylated hemoglobin, types of cataract and the situation of the patients'retina were collected, and the correlation to the expression of RAGE was further analyzed by the logistic regression analysis method.(2) Human lens epithelial cell (hLEC) line (SRA01/04) was cultured in vitro and treated by AGE-BSA with a series of concentration gradient (0 mg/ml, 0.25 mg/ml, 0.50 mg/ml and 1.00 mg/ml) in different experimental groups and by bovine serum albumin (BSA) treatment with similar concentrations as the control. After cocultivation with hLECs for 24 h, ROS content in all groups was detected with ROS detection kit and flow cytometry, ratio of GSSG/GSx with GSH and GSSG detection kit and colorimetric technique. In addition, RAGE expression was also examined using Western blot.(3) Human lens epithelial cell line (SRA01/04) was cultured in vitro and treated by AGE-BSA for a series of time period (0,2,8,16 and 24h) in different experimental groups and by BSA treatment for a similar time periods as the control. ROS content in all groups was detected with ROS detection kit and flow cytometry, ratio of GSSG/GSx with GSH and GSSG detection kit and colorimetric technique. In addition, RAGE expression was also examined using Western blot. Results(1)①The positive staining can be observed in cell membrane with different coloring gradient and an appearance of brown bulk particles in LECs of both diabetic cataract and age-related cataract. However, it did not present in the normal lenses. The expression of RAGE in epithelial cells of diabetic cataract lenses was significantly higher than that in age-related cataract and normal lenses (P < 0.0125); There was also a significant difference for RAGE expression in epithelial cells between age-related cataract and normal lenses (P<0.0125).②The correlation analysis indicated that the expression of RAGE had a positive correlation with patients'diabetes duration and their levels of glycosylated hemoglobin (P<0.05, OR>1). However, another clinical datum including sex, age, cataract types and situation of retina didn't have significant correlation with the RAGE expression (P>0.05).(2)①ROS content was increased in a concentration-dependent manner when cells were treated by AGEs in the experimental groups,whereas the similar changes did not present in the control groups. There were no significant differences of ROS content between the experimental groups and the control groups when the groups were treated with 0 mg/ml and 0.25 mg /ml AGEs and BSA, respectively. However, the significant differences of ROS content were observed when the experimental concentrations were up to 0.50 mg/ml and 1.00 mg/ml. Besides, GSSG/GSx ratio in the experimental groups also increased in a concentration-dependent manner. This ratio increased from 1.7±0.26% to 12.83±0.40% with the increase of AGEs from 0 mg/ml to 1.00 mg/ml. There were no significant differences of GSSG/GSx ratio between the experimental groups and the control groups when the groups were treated with 0 mg/ml and 0.25 mg /ml AGEs and BSA, respectively. However, the significant differences of GSSG/GSx ratio were observed when the experimental concentrations were up to 0.50 mg/ml and 1.00 mg /ml.②RAGE expression showed a tendency to increase in a concentration-dependentmanner when cells were treated by AGEs in the experimental groups. Compared with the control groups, the expression of RAGE protein in the experimental groups were increased significantly when cells were treated with 0.50 mg/ml and 1.00 mg /ml AGEs, suggesting that the expression of RAGE protein can be up-regulated by AGEs.③ROS content was increased in a time-dependent manner when the cells were treated by AGEs. At the ealier stage, the changes were not obvious. However, some significant changes can be observed when cells were treated after 16 h and 24 h. There were no significant changes of ROS content in the control groups at any time points. ROS content in the experimental groups were observed higher than those in the control group in 16 h and 24 h. In addition, GSSG/GSx ratio also increased in a time-dependent manner with AGEs treatment.This ratio increased from 0.63±0.15% to 10.63±0.40% from 0 h to 24 h AGEs treatment. GSSG/GSx ratio in the experimental groups increased very rapidly in the first 8 h while its increased tendency became mild in the next 16 h.④RAGE expression showed a tendency to increase in a time-dependent manner when cells were treated by AGEs in the experimental groups. Compared with the control group, the expression of RAGE protein in the experimental groups were increased significantly when cells were treated after 16 h and 24 h with AGEs, suggesting that the expression of RAGE protein can be up-regulated by AGEs.ConclusionRAGE was highly expressed in anterior capsules of human lens epithelial cells in diabetic cataract and age-related cataract patients, whereas its expression can not be observed in LECs of normal lenses. AGEs can change the redox state of LECs and up-regulate RAGE expression. Thus, the interaction of AGEs and RAGE may play an important role in the pathogenesis of diabetic cataract. |
PDF Full Text Request