Effect Of Thioltransferase On The Formation Of Diabetic Cataract Induced By High Glucose In Rat Lens

Purpose:Thioltransferase(TTase) is an important oxidoreductase in the lens. By restoring of the thiolated protein produced by oxidative damage, TTase plays a vital role in regulating the intracellular SH/-S-S-ratio, maintaining redox homeostasis in the lens. Through a variety of ways to induce oxidative stress, hyperglycemia of diabetes could result in diabetic cataract. To investigate the relationship between the role of of diabetic cataract and TTase, and to illustrate the mechanism of TTase in development of diabetic cataract, our study focused on diabetic cataract in vitro model, and investigated the effect of high glucose on the redox system of the cultured rat lenses in order to explore the protective effect of the lens that TTase redox system may occur. Methods:1. Lenses were incubated with different concentrations of glucose: control group(5.5mmol/L), high glucose groups(35.5, 65.5, 95.5mmol/L) and mannitol control group for 7 days. Following incubation, the lenses were evaluated daily using a dissecting microscope. After 7 days of incubation, lenses were homogenized in lysis buffer and processed for measurement the activities of TTase, catalase(CAT), superoxide dismutase(SOD) and the specific value of GSSG/T-GSH.2. According to the first part of results, the 35.5mmol/L glucose was selected as high glucose for rat lenses incubation. The incubated lenses were selected randomly at the time of 1d,3d,5d and 7d. The ratio of GSSG/T-GSH and the activity of TTase in each group were measured. TTase mRNA levels were detected by qRT-RCR. The TTase expression were observed in histological sections by HE and immunohistochemical staining at the time of 7d Results:1. The lenses treated with high glucose exhibited mistlike opacity in the early stages(P<0.05), while the lenses of control group remained relatively transparent. With the raise of the concentration of high glucose, the activity of TTase increased and the maximum appeared in 35.5mmol/L group(1.743±0.20 mU/mg protein P<0.01). No changes were observed in the mannitol control group(P>0.05). The specific value of GSSG/T-GSH of lenses with high glucose groups increased compared with the control group`s(P<0.05). There was no significant difference between each group treated with high glucose, and the difference was also no significant between the mannitol group and the control group. Compared with the control group, the activities of CAT and SOD decreased in the high glucose treatment groups(P<0.05). There was not significantly difference between each group treated with high glucose.2. HE staining and immunohistochemistry staining showed that lens fiber cells of high glucose group presented swelling, degeneration, vacuolar degeneration compared with the control. No significant difference was observed between the group treated with isotonic group. The ratio of GSSG/T-GSH of the group treated with high glucose was increased with prolonging the time of incubation. The ratio were 24.89±2.8%,35.99±2.49%,32.81±6.04% and 30.99±4.17% at the time of 1d, 3d, 5d and 7d, respectively. Compared with the control, the ratio were 2.21, 3.89, 2.38 and 2.74 times to the normal control group, respectively. The activities of TTase were gradually growing trend, showing as 1.25±0.31 mU/mg protein, 1.49±0.23mU/mg protein, 1.61±0.20 mU/mg protein and 1.91±0.28 mU/mg protein at the time of 1d, 3d, 5d and 7d, respectively. And compared with the corresponding period in the control, the activities were 1.58, 2.28, 2.21 and 2.21 times to the normal control group, there were statistically significant differences. In high glucose group, the levels of TTase mRNA gradually increased.and reach the top at 5d, then it decreased. In 35.5 mmol/L high glucose group, the levels of TTase mRNA was 2.44 times to the normal control group at the time of 1d(P>0.05), Then, the levels of TTase mRNA were suddenly fell slightly after rose gradually. The levels of TTase mRNA were 6.58, 6.78 and 3.88 times to the normal control group, respectively(P<0.05). No significant difference was observed between the group treated with isotonic group. Conclusions:1. The antioxidant system of the lens was activated by the high glucose following the increased activity of TTase, the specific value of GSSG/T-GSH, and the decreased the activities of CAT and SOD. The gradually decreased oxidation resistance in the lens may contribute to the development of diabetic cataract. TTase may be involved against high glucose-induced oxidative stress and plays against oxidative stress, regulating the oxidation-reduction lens steady protection.2. No direct correlation had found between the morphological changes of diabetic cataract induced by high glucose and expression of TTase protein. But there was a significant increased activity at the early stage. Reflected antioxidant statuses of GSSG/T-GSH were increased in the corresponding period. Detected by qRT-RCR, the levels of TTase mRNA were confirmed that prompt response of TTase may play an important protective role in the early stages of diabetic cataract, thus delaying the development of cataract.