The Effect Of N-acetylcysteine On Diabetic Cataract In Animal And Cytobiology Study

Purpose: To evaluate the effect of N-acetylcysteine (NAC) eye-drops on the progression in diabetic cataract formation induced by streptozotocin (STZ), and the effect of NAC on apoptosis of human lens epithelial cells (hLEC) induced by high glucose.Methods:(1) Eighty-five Sprague-Dawley (SD) rats were selected and diabetes was induced by single intraperitoneally injection of 1% streptozotocin (65 mg/kg body weight). The control group (normal group,Group A) received only sodium phosphate buffer drop (25mmol/L,pH7.4). Then 59 rats with the random blood glucose above 14mmol/L were divided into three groups (group B-D). Group D animals received sodium phosphate buffer drop (25mmol/L,pH 7.4), and those in the groups B and C received 0.01%NAC and 0.05% NAC drops, respectively.(2) Lens transparency was monitored by slit lamp bio-microscope and the opacities of the lenses were classified into six stages according to the Oxford system. At the end of 4 weeks, 8 weeks and 13 weeks, the animals were killed and the level of glutathione (GSH), the activities of glutathione reductase (GR) and catalase (CAT), the level of the advanced glycation endoproducts (AGEs) were investigated in the lenses extracts. Blood glucose, urine glucose and body weight were also determined.(3) Human lens epithelial cells were divided into three groups: the normal group (DMEM), the treated group (DMEM+NAC+HG), and the untreated group (DMEM+HG). Apoptosis of hLECs was induced by 120 mM high glucose (HG), and the protection of NAC on apoptosis were analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The activation of apoptotic stress-signaling proteins (Caspase-3) was observed by spectrophotometry.Results:(1) About 79%(59/75) of the rats responded to the STZ injection (blood glucose> 14 mmol/L). One month after the diabetic model was set up, three rats were rejected for the decrease of blood glucose (<14 mmol/L). There were statistically significant higher of blood glucose (P<0.05) and lower of the body weight (P<0.05) in the diabetic rats than that of normal rats throughtout the experiment period.(2) The opacity of lenses in diabetic untreated group (group D) was not observed until the 3rd week after STZ injection. The progression in lens opacity induced by diabetes showed a biphasic pattern in which an initial slow increase in the first 6 weeks was followed by a rapid increase in the next 7 weeks. The significant opacity of the lenses from the two NAC eye-drops treated groups was not noticed until the 4th week after STZ injection. However, the progression of lens opacity in the two NAC treated groups was slower than that of the untreated group in the earlier five weeks, and especially in the 4th week. There was statistically significant difference in lens opacification between the 0.01% NAC treated group (P=0.023,P<0.05) and the untreated group. However, no statistically significant difference was noticed in the 0.05% NAC treated group(P>0.05) compared with the untreated group, although the slower progression of lens opacity was observed in this group. Moreover, there was no statistically significant difference in the progression of lens opacification between the two NAC treated groups.(3) A decrease of GSH, inactivation of GR and CAT was found in the lenses extracts of the diabetic rats at 4th, 8th, 13th week after STZ injection. Expecially in the 13th week, the content of GSH was decreased by 50%(P<0.05), the inactivation of GR and CAT was reduced by 20%(P<0.05)and 50%(P<0.05). The diabetes resulted in an increase level of AGEs in diabetic rats lenses during the whole process, with the increase by 46.1%(P<0.05)of AGEs compared with the normal group at the 13th week. While the levels of GSH and CAT activity were both higher in the two NAC treated groups compared with the untreated group at every observation time. However, there was no statistically significant difference (P>0.05) between these groups. At the 4th week, the contents of GSH in 0.01% NAC treated group and 0.05% NAC treated group were increased by 10.0% (P>0.05) and 20.4%(P>0.05);the activity of CAT was increased by 10.6% (P>0.05) and 13.3% (P>0.05), respectively. Moreover, no statistically significant difference was observed in the activity of GR and the content of AGEs between the treated groups and the untreated group. Although there was no statistically significant in the biochemical evaluation comparing the two NAC treated groups with the untreated group, the increase of GSH level and the activity of CAT during the whole experiment were still obvious.(4) NAC can inhibit the apoptosis of HG induced hLEC. The hLECs were induced apoptosis in a time and a dose dependent manner within a certain range of treating time and dose. The apoptosis rate of the untreated group was significantly higher than that of the NAC treated group at 72 h, (34.14±1.98)% vs (46.37±2.08)% (P<0.05). TUNEL-positive cells were observed chromatin condensation, marginate in crescents around the nuclear, nuclear pyknosis, and the nuclear became buffy color. Caspase-3 was activated with the treatment of HG. NAC inhibited apoptosis and Caspase-3 activation induced by HG. The activation of Caspase-3 was decreased by 30.1% (P<0.05) at 48 h and 39.0% (P<0.05) at 72 h in the NAC treated group.Conclusion:(1) NAC eye-drops can slightly inhibit the progression of the diabetic cataract at the earlier stage. It may maintain the lens transparency and function by serving as a precursor for glutathione biosynthesis.(2) NAC prevents apoptosis of human lens epithelial cells possibly through regulation of the activity of Caspase-3.(3) NAC delays the progression of diabetic cataract possibly by inhibition the apoptosis of hLEC, and increasing the content of GSH in lens, then prevention the oxidative damage.