| Objective:Persistent hyperglycemia increases the formation of advanced glycation end products(AGEs). Oxidative stress induced by AGEs has been demonstrated to stimulate formation of diabetic cataract. Thioltransferase(TTase) is a member of the thiol-disulfide oxidoreductase enzyme family. In this study, we examined the effects of AGEs on oxidative state. We also investigated the expression of TTase and its activity. Furthermore, we explored the possible mechanism of TTase to counteract the oxidative stress induced by AGEs. Our study provides a novel understanding of pathological mechanism of diabetic cataract.Methods:1. HLE B3 were treated with 1.5 mg/ml AGEs-BSA as the experimental groups(AGEs-BSA group) and cells were treated with bovine serum albumin(BSA) as negative control(BSA group). Cells were collected at the time point of 1 d, 2 d, 3 d and 4 d. Then the TTase activity, reactive oxygen species(ROS) levels, superoxide dismutase(SOD) and catalase(CAT) activity were measured. TTase m RNA levels were detected by q RT-RCR and its protein level was detected by Western blot.2. Then si RNA was used to knockdown the expression of TTase. We accessed the activity of CAT and SOD, the content of ROS and the ratio of GSSG/T-GSH at the time point of 2 d in different groups, respectively.Results:1. ROS content increased in a time-dependent manner in AGEs-BSA group. The specific content of ROS in this group were 153.67±24.57, 180±11.55, 268.01±20.50 and 313.33±35.46 relative fluorescence units from 1 d to 4 d. There was statistically significant difference of ROS content between the AGEs-BSA group and the normal group(P<0.05). CAT activities in AGEs-BSA groups were 10.07±0.84, 9.27±0.72, 7.05±0.88 and 5.30±1.67 U/mg protein from 1 d to 4 d, which decreased obviously compared with the normal group(P<0.05). SOD activities were 104.21±3.95, 92.98±8.98, 86.54±3.57 and 81.56±5.06 U/mg protein from 1 d to 4 d. CAT and SOD activities decreased in a time-dependent manner in the treatment groups.2. The TTase m RNA increased after treatment with AGEs-BSA for 1 d, peaked at 2 d, and then decreased gradually. The difference in the TTase m RNA level between the AGEs-BSA and the control group was statistically significant at 2 d(P<0.01). The activities of TTase were 8.03±0.86, 10.82±0.51, 11.05±0.83 and 8.63±0.77 m U/mg protein from 1 d and 4 d, respectively. The differences in the TTase activities between the AGEs-BSA and the control group was statistically significant at 2 d and 3d(P<0.05). In AGEs-BSA group, the protein level of TTase increased gradually from 1 d to 4 d. From the third day, the protein level of TTase was remarkably up-regulated in the AGEs-BSA groups compared with the controls(P<0.01).3. In this part, this test was divided into four different groups: control group, AGEs-BSA group, si RNA negative control group and si RNA + AGEs-BSA group. The content of ROS in each group were 93.33±21.85, 190.30±9.84, 188.02±19.31, 280.32±18.35 relative fluorescence units, respectively. The difference in ROS level between the si RNA + AGEs-BSA and the si RNA negative control group was statistically significant(P<0.05). And the ratio of GSSG/T-GSH in each group were(3.20±0.47)%,(11.3497±0.62)%,(10.86±1.50)%,(16.45±0.64)%, respectively. There was statistically significant differences of GSSG/T-GSH ratio between the si RNA + AGEs-BSA and the si RNA negative control group(P<0.05). Moreover, the activities of CAT and SOD was significantly down-regulated in the si RNA + AGEs-BSA group compared with the si RNA negative control group(P<0.05).Conclusions:AGEs induce oxidative stress and change the redox state in human lens epithelial cells since it increase ROS content and impair activities of SOD and CAT. TTase m RNA level and its activity are up-regulated by AGEs. And this upregulating in the early stage may be involved in counteracting oxidative stress induced by AGEs. TTase plays a significant role in neutralizing oxidative stress. And the mechanism of TTase may be through decreasing the specific value of GSSG/T-GSH, repairing the activities of CAT and SOD, and then decreasing ROS content. |
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