Study On Protective Effect Of Recombination Ribonulease Inhibitor On Cataract Of Rat

Human ribonuclease inhibitor (hRI), an acidic protein (pI=4.7), is ubiquitous in the cytoplasm of human cells with molecular weight of about 50kDa, and its gene locates in No.11 chromosome (11p15.5). The activity of ribonuclease A (RNase A) can been inhibited by hRI through tight combination with it. Its three-dimensional structure is charactered by alternating units ofα-helix andβ-strand that form a striking horseshoe shape. The hRI possesses a highly repetitive amino acid sequence that is rich in leucine residues. These leucine-rich repeats (LRRs) are present in a large family of proteins that are distinguished by their display of vast surface to foste protein-protein interactions. The unique structure and function of hRI have resulted in its emergence as the central protein in the study of LRRs. The hRI consists of 32 highly conserved cysteine residues. At least 30 cysteine residues could exist with reduced thiol groups which is necessary for hRI to maintain its biological activities. High content of cyeteine residues maybe make hRI with antioxidation.During the pathogenesis of diabetic cataract , a large quantity of free radicals produced in the process of reduced monosaccaride oxidation damages proteins and membranes of len epithelial cells. Degenerated proteins and damaged cells lead to genesis and development of diabetic cataract. We had found that rat glial cell line C6 transfected with hRI cDNA can resist more injuries with H₂O₂ than the control C6 cells.In this experiment, the diabetic cataract model was established by injecting intraperitoneally streptozotocin into Wistar Rat. Rosetta transformed with the fusion expressive plasmid pGEX-6p-1-ri were cultured and collected by centrifugation.The precipitated Rosetta were crushed with hypersound and centrifugation. The supernatant was loaded on Glutathione-Sepharose 4B affinity chromatography column to extract and purify the fusion protein GST-hRI. The hRI was applied to treat the diabetic cataract animal model of Wistar Rat to observe its protective effect on diabeticcataract. The result indicated that GST-hRI could to slow down the genesis and development of diabetic cataract in a dose-dependent way. The statistical analysis of biochemical indexes shown that was GSH content and SOD activity of high-dose GST-RI therapeutic group obviously higher than those of other groups, except the normal group. MDA content of high-dose GST-RI therapeutic group was less than that of other groups, and GSH-PX activity same as other therapeutic groups. We concluded that GST-hRI relieve the oxidative stress in lens. Recombinant hRI could be prevented and therapied rat diabetic cataract induced by streptozotocin.Conclusion:1. Rat diabetic cataract model was successfully established by intraperitoneal injection of streptozotocin.2. GST-hRI has the protective effect on the genesis and development of diabetic cataract induced by streptozotocin in a dose-dependent way. This effect of GST-hRI is superior to Baineiting.3. This study provides given experimental basis for farther application of recombinant hRI. Recombinant hRI maybe become an effective new drug to treat cataract.