| Objective:Thioltransferase (TTase) is a small cytosolic protein with low molecular weight. Itcatalyzes specifically the reduction of proteins that are thiolated by GSH and plays a vitalrole in maintaining redox homeostasis in the lens. High glucose can induce oxidativestress through a variety of ways, eventually leading to the formation of diabetic cataract.This study aimed to investigate the expression and activity of TTase in human lensepithelial cells (HLE B3) incubated with high glucose, and to explore the possible role ofTTase in the fight against high glucose-induced oxidative stress, which may lay a solidfoundation for elucidating the interaction between glycosylation and oxidative damage.The first part of the experiment was to observe the expression and activity of TTaseincubated with different concentrations of glucose in human lens epithelial cells with time.In the second part, we knocked down TTase by using RNA interference techniques. Thereactive oxygen species (ROS), GSSG content and superoxide dismutase (SOD), catalase(CAT) activity in human lens epithelial cells were measured and cell apoptosis rates were detected under joint effect of TTase RNAi and high glucose to explore the possible role ofTTase.Methods:1．Human lens epithelial cells were cultured in DMEM containing10%(v/v) fetalbovine serum and different concentrations of glucose (5.5mmol/L as the normal controlgroup,25.5mmol/L,35.5mmol/L and45.5mmol/L as the high glucose groups). Mannitolhypertonic control groups were set accordingly to exclude the effects of osmotic stress.Cells were collected at the time point of1d,2d,3d and4d, and total RNA wasextracted. TTase mRNA levels were detected by qRT-RCR. According to the first set ofresults,35.5mmol/L as the high glucose concentration was selected throughout the rest ofexperiments. TTase expression was detected by Western blot and its activity was measuredfrom1d to4d under35.5mmol/L high glucose concentration.2．Human lens epithelial cells were transfected with TTase siRNA and the efficiencyof transfection and interference were detected. The optimal siRNA was screened for thefollowing experiments. Normal control group, high glucose group, TTase RNAinterference (RNAi) group and TTase RNAi+high glucose group were set. TTase activity,ROS levels, GSSG/T-GSH, SOD and CAT activity were measured and cell apoptosis rateswere detected at the time point of3d.Results:1．In high glucose group, the levels of TTase mRNA gradually increased and reachthe top at2d, then it decreased to the normal level at4d. In25.5mmol/L high glucosegroup, the levels of TTase mRNA from1d to3d were2.53,6.06,3.33times to the normalcontrol group, respectively (P<0.05), and returned to the normal level at4d (P>0.05). In35.5mmol/L high glucose group, the levels of TTase mRNA from1d to3d were6.24,9.28,3.12times to the normal control group respectively (P<0.05) and returned to thenormal level at4d (P>0.05). In45.5mmol/L high glucose group, although the levels ofTTase mRNA from1d to3d were higher than that of the normal control group, the differences were statistically significant only at2d, nearly6.01times to the normalcontrol group (P<0.05). The levels of TTase mRNA returned to the normal level at4d(P>0.05). There were no significant differences of TTase mRNA levels in mannitolhypertonic group compared with the normal control group (P>0.05). The TTase activity inthe normal control group was6.69±0.47mU/mg protein. In35.5mmol/L high glucosegroup, the TTase expression gradually increased from1d to4d detected by Western blot.TTase activities were8.37±1.24,10.19±0.66,11.23±1.75,10.50±0.70mU/mg proteinfrom1d to4d, respectively, which indicated that the TTase activity gradually increasedfrom1d to3d followed by a decrease. There were statistically significant differencesfrom2d to4d compared with the normal control group (P<0.05).2．The efficiency of transfection was about81%, which was detected by flowcytometry. The optimal siRNA was screened according to the efficiency of interference.The optimal siRNA down-regulate TTase expression to39.5%of the normal control group(P<0.05). The TTase activities were6.68±0.47,11.23±1.75,3.58±0.70,5.49±1.15mU/mg protein in the normal control group, high glucose group, TTase RNAi group andTTase RNAi+high glucose group respectively. In high glucose group, TTase activityincreased (P<0.01) whereas TTase activity decreased (P<0.05) in TTase RNAi group.There were no significant differences between TTase RNAi+high glucose group and thenormal control group (P>0.05). The content of ROS were155.70±11.50,186.33±11.59,219.00±11.00,265.33±16.50relative fluorescence units in the normal control group,high glucose group, TTase RNAi group and TTase RNAi+high glucose grouprespectively. RNAi+high glucose group possessed the highest ROS levels, which wassignificantly higher than that of other groups (P<0.05). The ratio of GSSG/T-GSH were(3.81±1.08)%,(8.97±0.95)%,(10.96±1.28)%,(14.15±1.48)%in the normal controlgroup, high glucose group, TTase RNAi group and TTase RNAi+high glucose group,respectively. The highest ratio was found in RNAi+high glucose group, which wassignificantly higher than that of other groups (P <0.05). The activities of SOD and CATwere also measured in the four groups. The lowest SOD and CAT activities (47.15±5.41U/mg protein and4.46±0.74U/mg protein) were found in TTase RNAi+high glucose group (P<0.05). However, no obviously cell apoptosis was obsevered among the fourgroups, and no significant differences were showed in cell apoptosis rates (P>0.05).Conclusions:High glucose can increase ROS content in human lens epithelial cells, therefore itinduce oxidative stress, which resulted in the decreased GSH content and increased GSSGcontent, impaired activity of SOD and CAT. High glucose can promote the expression ofTTase and enhance the activity of TTase, which might be an adaptation response tooxidative stress induced by high glucose. The mechanism of its protective role might exertthrough increasing GSH content, decreasing GSSG content, repairing the impaird SODand CAT, eventually leading to the decreased ROS content and thus sustaining redoxhomeostasis in the cells. |
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