The Role Of Thioltransferase In The Formation Of Diabetic Cataract

Purpose: Thioltransferase(TTase) plays an important role in regulating the redox homeostasis in the lens. The purpose of this study is to investigate the function of TTase anti-oxidant enzyme system in the formation of diabetic cataract. Therefore, three parts of experiments were designed. The expression and activity of TTase were measured and its relationship with the biochemical changes and clinical informations were analyzed in the anterior capsule of diabetic cataract and age-related cataract. Streptozotocin(STZ) induced diabetic cataract model were established in rat and the progression of lens opacification at diferrent age were observed. The expression and activity of TTase were examined and the relationships with the biochemical changes and lens opacification level were analyzed in rat lenses. To investigate the possible role of TTase in high glucose-induced oxidative damage and apoptosis in human lens epithelium cells(HLECs). The expression and activity of TTase were examed in different glucose concentration induced HLECs at several differenttime.Methods: 1.The anterior capsules of 108 diabetic cataract and age-related cataract patients were collected during phacoemulsification procedures. Then they were divided into three groups by age: 40 to 55, 56 to 65 and 66 to 80 years to detect the expression and activity of TTase, GSH and GSSG levels. The relationships of TTase activity with such clinical informations as patients’ age, duration of diabetes, fasting plasma glucose, visual acuity, glycosylated hemoglobin and GSH were analyzed. And the expression and activity of TTase were also measured in anterior capsules of 96 different types of cortical, nuclear and mixed age-related cataract patients aged 50 to 60 years. 2.Thirty male SD rats were randomly divided into two groups. DC group received a single intraperitoneal injection of streptozotocin(STZ, Sigma Company, 65 mg/kg). The control group was given citrate buffer alone. Rats with blood glucose levels greater than 16.7 mmol/L 72 h after STZ injection were considered as diabetic rats. The body weight and the blood glucose of rats were measured regularly after STZ injection. After dilation of the pupils with 1% compound tropicamide eye drops, the progression of cataract formation in the lens of all rats was observed and noted weekly by slit lamp. The grading of lens opacification was performed according to the Oxford University system. After 12 weeks, the rats were sacrificed and the lenses were dissected out, then placed into Eppendorf tubes and frozen at-80℃ until analyzed. The expression and activity of TTase were examined and the levels of glutathione and GSSG were measured by DTNB coloration measurement. The relationships between the activity of TTase and the biochemical changes and lens opacification level were analyzed using SPSS statistical analysis software. 3. Human lens epithelium cells(HLE B3) were incubated with different concentrations of glucose: control group(5.5m M), high glucose groups(15.5-35.5m M) and mannitol control group(35.5m M, to exclude the influence of osmotic pressure). After treated with different concentrations of high glucose for 24 hours, HLECs were gathered and TTase expressions were examed to establish a concentration curve.According to the result of concentration curve, HLECs were gathered at 0h,2h,8h,16 h and 24 h after treated with high glucose of 35.5m M, respectively. TTase expressions were examed. The levels of glutathione and GSSG of HLECs were measured. The induction of apoptosis was measured by flow cytometer(FCM). Western blot analysis was performed to measure the expression of apoptosis associated protein Bax and Pro-Caspase-3.Results: 1.The expression and activity of TTase(2.64±0.83 m U/mg protein) in anterior capsules of 40 to 55 years group of diabetic cataract patients were significantly higher than that of age-related cataract patients(2.36±0.78 m U/mg protein)(P<0.05). However, no significant difference between older than 56 years groups were found. Correlation analysis showed that TTase expression and activity were correlated with diabetic patients’ age(R=-0.755), duration of diabetes(R=-0.515), glycosylated hemoglobin(R=-0.607), the degree of proliferative diabetic retinopathy(R=-0.438), GSH(R=0.730) and GSSG(R=-0.438), and were not associated with fasting plasma glucose(R=-0.147), visual acuity(R=0.199), the different types of cataract(R=-0.134). The differences of activity of TTase were not significant in cataract lens capsule of cortical(2.24 ± 0.87 m U/mg protein), nuclear(2.14 ± 0.61 m U/mg protein) and mixed age-related cataract(2.09 ± 0.76 m U/mg protein). 2.After injection of STZ 72 h the blood glucose level was significantly higher in all of the diabetic rats compared with the normal group(P<0.05). After dilation of the pupils with 1% compound tropicamide eye drops, the opacity of lenses in diabetic rats was observed in the 4th week after STZ injection by slit lamp. Then the lenses opacification progressed slowly in the next 4 weeks, followed by a rapid increase in the next 4 weeks. Lens opacities were consistently severer in the diabetic group compared with the control group. The lenses remained transparency in the control group throughout all the experiment peroid. After 12 weeks the glutathione level in the diabetic group(28.31±4.76 umol/g protein)was decreased by 50% compared with that of the normal rats(52.97±6.44 umol/g protein)(P<0.05). GSSG level in the diabetic group(6.94±1.40 umol/g protein) increased compared with that of the normal rats(2.03±0.45 umol/g protein)(P<0.05). The expression and activity of TTase in the lenes of diabetic cataract rats(0.81±0.27U/g protein)were significantly lower than that of the control group(1.26±0.29U/g protein)(P<0.05). Correlation analysis showed that TTase expression and activity in the diabetic rats were correlated with the degree of the lenses opacification and the changes of GSH(R=0.593,P<0.05)and GSSG levels(R=-0.541,P<0.05), respectively. 3.The expression of TTase increased gradually with the raise of the concentration of high glucose and the maximum expression appeared in 35.5m M group. No changes were observed in the osmotic pressure control of mannitol group. The expression of TTase increased after treated with high glucose of 35.5m M for 2 hours(5.41±0.91 m U/mg protein)and the maximum expression appeared at 8 hours(9.47±1.22 m U/mg protein)which was much higher(P<0.05) than that of the control group(4.83±0.62 m U/mg protein). Then it decreased gradually. The GSH level decreased after treated with high glucose of 35.5m M and was significantly lower at 16 hours(7.41±0.92 nmol/mg protein) than that of 0h(10.33±1.56nmol/mg protein)(P<0.05). The GSSG(0.78±0.082 nmol/mg protein) and GSSG/GSH(6.38±0.72%) level increased after treated with high glucose of 35.5m M for 2 hours and the raised at 8 hours(P<0.05). The GSSG level raised to 1.23±0.15nmol/mg protein and GSSG/T-GSH level was 12.1±1.49%(P<0.05)with high glucose of 35.5m M for 24 hours. No significant increase in apoptosis rate was observed between the control group and the high glucose group after treated with high glucose of 35.5m M for 24 hours. The expression of apoptosis associated protein Bax increased and Pro-Caspase 3 decreased from 24 hours after treated with high glucose of 35.5m M(P<0.05).Conclusions: 1.TTase activity was correlated with diabetic patients’ age, duration of diabetes,glycosylated hemoglobin, the degree of proliferative diabetic retinopathy, GSH and GSSG. It may play an important role in the formation of diabetic cataract. The correlation between the formation of different types of age-related cataract and TTase were not obvious. 2.The intraperitoneal injection of streptozotocin can induce rats of diabetes with an apparente lens opacification. TTase expression and activity in the diabetic rats decreased in diabetic rats. The GSH levels decreased and GSSG levels increased. And the TTase activity was correlated with the degree of the lens opacification and the GSH, GSSG levels. It may have a protect function in the formation of diabetic cataract and oxidative damage. 3.The expression of TTase in HLECs increased after treatment with high glucose and it showed a concentration-dependent manner. At the same time, the GSH level decreased and GSSG level increased. The expression of apoptosis associated protein changed, indicating that TTase might participate in the cell reaction of high glucose stimulus. We assume that TTase is associated with the high glucose induced oxidative damage and apoptosis of HLECs. This may have a protective effect of HLECs against oxidative insult.