| Objective:1. To set up the diabetic cataract animal model and to study the mechanism of diabetic cataract in rats.2. To explore the effect and mechanism of glutathione ethyl ester on streptozotocin–induced diabetic cataract.3. To primarily investigate the effect and the mechanism of carnosine on human lens epithelial cells apoptosis induced by high glucose.Methods:The lens transparency was detected in sixty-six male Sprague-Dawley (SD) rats. Ten healthy rats of the same age were taken out randomly as normal animals (Group A), receiving 0.02 mol/L citrate buffer (65mg/kg, pH 4.5) alone. The rest 56 rats were randomly divided into the glutathione ethyl ester (GSH-EE) treated group (Group B) and the diabetic group (Group C). Diabetes was induced by streptozotocin (65 mg/kg) intraperitoneally. Rats with blood glucose levels greater than 14mmol/L on 72 h after STZ injection were deemed diabetic rats. Five days before STZ injection the rats in group A and C received 25 mmol/L phosphate buffered solution (pH 7.4), and those in groups B received 0.1% solution of GSH-EE (pH 7.4), twice daily, respectively. Blood glucose was measured monthly, and urine glucose and body weight were also determined weekly. Cataract progression due to hyperglycemia was monitored by slit lamp biomicroscope weekly according to the Oxford University System. At the end of 4 wk, 8 wk and 13 wk after injection, the rats were killed and the lenses were dissected out. The level of glutathione (GSH), the activities of glutathione reductase (GR) and catalase (CAT), and the level of advanced glycation end products (AGEs) were determined in the lenses extracts.The apoptotic human lens epithelial cells were induced by high glucose, and then these cells were treated with carnosine at different concentrations. The concentrations of high glucose and carnosine were tested by MTT assay. Human lens epithelial cells were divided into three groups and cultured in vitro: Normal group (DMEM), High glucose group (DMEM + 120 mmol/L high glucose), Carnosine-treated group (DMEM + 120 mmol/L high glucose +25 mmol/L carnosine). Following incubation, human lens epithelial cells from each group were lyzed and the activity of Caspase-3 from supernatants was measured after 24,48, 72 and 96 hours incubation by means of spectrophotometry, respectively. Apoptotic lens epithelial cells were assayed by using TUNEL method and apoptosis rate of lens epithelial cells was calculated under light microscope.Results:1. Blood glucose were significantly greater in all of the diabetic rats on 72 h after injection of STZ (29.43±5.23 mmol/L, P<0.01) compared with the normal group (6.99±0.42 mmol/L). Throughout the study period, elevated blood glucose and decreased body weight were observed in the diabetic rats.2. The opacity of the lenses in the diabetic untreated group wasn't observed until the 3 wk after STZ injection. Lens opacification progressed in a biphasic manner, an initial slow increase during the 3~6 wk of diabetes followed by a steep increase in the 7~13 wk.3. The opacity of the lenses in the GSH-EE treated group wasn't noticed until the 4 wk after STZ injection. There was statistically significant difference in the lens opacification between the treated group and the untreated group at the 4 wk after STZ injection (P<0.05). No statistically significant difference was obsevered after the 5 week in the lens opacification between the two groups and the progression of lens opacification in all diabetic groups became aggravated.4. Compared with normal group, the level of GSH, and the activity of glutathione reductase (GR) and catalase (CAT) were decreased in the lenses extracts of the diabetic rats after STZ injection. The content of GSH was greater in the GSH-EE treated group than that of the diabetic untreated group (mg/gprot, 4 wk: 80.32±10.02 vs 68.44±9.05; 8 wk: 56.62±9.82 vs 50.27±8.43) at the 4 and 8 wk after STZ injection. Although there was an increase of 17.5% (at the 4 wk) and 12.6% (at the 8 wk) comparing with the two groups, there was still no statistically significant difference between these two groups at the 4 and 8 wk (P>0.05), respectively. Moreover, no statistically significant difference between the GSH-EE treated group and the diabetic untreated group was observed in the activity of catalase and glutathione reductase. Again no statistically significant difference between the two groups was noticed in the level of AGEs. It showed GSH-EE could prevent the decrease of GSH. 5. Treatment with different concentrations of carnosine (10, 25, 50 mmol/L) increased the survival rate of human lens epithelial cells. Then the dose of 25 mmol/L carnosine was selected at the survival rate around 50%.6. Treatment with 25 mmol/L carnosine decreased the activity of Caspase-3. The activity of Caspase-3 was decreased by approx. 11% (P>0.05), 22% (P<0. 05), 8% (P>0.05) in the carnosine-treated group at 48, 72, 96 hours, respectively, compared with the high glucose-induced group.7. The TUNEL results showed that the percentage of apoptotic cells was gradually increased with the prolonged incubation time. There was a decrease by approx. 4% (P>0.05), 13% (P<0. 05), 5% (P>0.05) of the epithelial cell apoptosis rate in the carnosine-treated group at 48, 72, 96 hours, respectively, compared with high glucose-induced group.Conclusions:1. The diabetic model induced by STZ intraperitoneal injection is reliable for the study in diabetic cataract. A single intraperitoneal injection of streptozotocin (65 mg/kg) can induce diabetes with a long-term, stable hyperglycemia in adult rats. The early character of the diabetic cataract displays clearly in this model at 4 wk after STZ injection.2. GSH-EE eye drop is safe for the diabetic rats and it can inhibit the progression of diabetic cataract at the earlier stage. Its function weakens during the intermediate and advanced stages in diabetes. It may maintain the lens transparency and function by serving as a precursor for glutathione biosynthesis.3. High glucose can cause human lens epithelial cells apoptosis by activating Caspase-3. Carnosine can inhibit the decreased survival rate of human lens epithelial cells induced by high glucose and the activity of Caspase-3, and thus prevent human lens epithelial cells from apoptosis.
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