Comparative Proteomic Analysis Of Diabetic Rat Retinae

Objective:To observe the pathological changes of retinal tissues in diabetic andnormal rats, establish and optimize a two-dimensional polyacrylamidegel electrophoresis (2-DE) technique for retina pro.teomics to improvethe resolution and reproducibility.To evaluate the application of two-dimensional electrophoresis andmass spectrometry in the research ofproteins expressed in retinas.To compare and identify the differences in expressed proteins betweennormal and diabetic rat retinas, and to .analyze the molecularoathogenetic mechanisms of diabetic retinopathy. differential expressions were obtained through analysis of 2-DEimages with PDQuest 7.3.1 software.protein spots that exhibit statistically significant variations (P＜0.05)were cut from gels, then the proteins were digested in-gel by enzymeand the generated peptides were identified by tandem massspectrometry (MS/MS).These proteins were analyzed through database searching andbioinformatics.Results:No obvious morphological change in retinas of 8 weeks diabetic rats wasobserved in comparison with normal tissues.By choosing appropriate sample amount, using pre-cast IPG dry strips (pH 3-10, pH5-8) and casting 12％equal gel, satisfactory 2-DEmaps of retina were obtained. A steady 2-DE technique wasestablished.Through 2-DE, we detected 36 spots in 2-D gel of diabetic retinas thatexhibit statistically significant variations(P＜0.05), includingup-regulation of 5 proteins in diabetic rat retinas, down-regulation of23, and disappearance of 8, in comparison with normal tissues.Crystallin alpha B is one of small heat-shock proteins (sHSPs) which arecytoprotective molecular chaperone, plays an important role in the protectionand modulation of the cytoskeleton. It has anti-apoptotic functions byinterfering with the activity of various apoptotic proteins. Our findingsdemonstrate for the first time that crystalsin alpha B was higher expressed inretinas of diabetic rats than those of normal rats, which weakens the retinaltissue damage through cytoprotective action. It may be regarded as a powerfulbiochemical marker for diagnosis, treatment and prognosis of DR.Albumin is the most abundant transport and depot protein in the mammalian vasculature, regulates the colloidal osmotic pressure of blood, increases theinternal diameter of blood vessels, especially arterioles or capillaries. Albuminmay play a role in nitric oxide signaling through mediating formation ofS-nitrosothiols (RS-NO). Albumin has antioxidant activity, which may inhibitthe reactions brought about by dioxygen (02) or peroxides, low-expression ofalbumin in retinas affects blood colloidal osmotic pressure and nitric oxidesignaling, increases vascular resistance and permeability, and at last, aggravatesratinal cell death and causes blindness.Dimethylarginine dimethylaminohydrolase 2 (DDAH II) belongs to the DDAHfamily and has dimethylargininase activity which hydrolyzes N(G),N(G)-dimethyl-L-arginine (asymmetrical dimethylarginine; ADMA) and N(G)-monomethyl-L-arginine (MMA). ADMA and MMA act as inhibitors ofNO synthases (NOS), so DDAHⅡplays an important role in nitric oxidegeneration. Hyperglycemia increases NO production in retinal and vitreoushumor cells, Excessive production of NO causes cell death and is a hallmark ofinflammatory diseases. Down-regulated expression of DHHGGKK in the retina ofdiabetic rat results in the increase of plasma ADMA and decrease of NOsynthesis. So reduction of DDAHⅠin diabetic retinas may protect the retinalcells from injury induced by hyperglycemia.Triosephosphate isomerase 1 (TIM-1) plays an important role in glycolysis bycatalysing the reversible interconversion of D-glyceraldehyde 3-phosphate (GAP)and dihydroxyacetone phosphate (DHAP). Glucose is broken down to pyruvatevia a chemical pathway that involves 10 enzymes and TIM-1 is the fifth enzymein this reaction chain that converts DHAP into GAP. Hyperglycemia appears toincrease nitration of glycolytic enzymes in retina and retinal cells. High bloodglucose level of diabetic rats may increase nitration of TIM-1, which leads tolow expression of TIM-1 and decreases the synthesis of ATP. It is possible toregard TIM-1 as an effective marker for diagnose or treatmentfor DR.Guanylate kinase 1(predicted) (GK-1) catalyzes the TP-dependent phosphorylation of GMP into GDP, and is essential for recycling GMP andindirectly, cGMP. In the retina of diabetic rat, low expression of GK-1 reducesthe phosphorylation of GMP into GDP, which increases GMP level. Thenincreased GMP level inhibits the hydrolyzing cGMP to GMP and results in theaccumulation of cGMP, which does not close the cation-specific channels. As aresult, the increase of cytosolic calcium level interrupts hyperpolarization of theplasma membrane and generation of neural signal. Maybe, it is one of themechanisms of blindness in diabetic patients with retinopathy.ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d .(ATPsynthase subunit d) catalyzes ATP synthesis from ADP and phosphate utilizingthe energy derived from an electrochemical gradient of protons across the innermembrane during oxidative phosphorylation. The exact function of ATP synthasesubunit d is unknown. Down-regulated expression of ATP synthase subunit d inthe retinas of 8-weeks diabetic rats may reduce ATP synthesis in retinas,combined with affecting the function of retina cells.Peroxiredoxin 6 (Prdx 6) is a unique, bifunctional protein with both GSHperoxidase and phospholipase A2 activities, which protect cells from oxidativestress damage by reducing cellular peroxides. In the retina of the diabetic rat,disappearance of Prdx 6 contributes to accumulation of cellularperoxides-convert, and accentuates the tissue oxidative injury.Predicted crystallin beta A1 have previously been associated with elevatedintraocular pressure (IOP), however, the functions of crystallin beta A1 innon-lenticular tissues remains equivocal so far.Conclusions:The established 2-DE technique of retina proteins can be effectivelyapplied in proteomics of retina diseases.The differences of protein expression were observed in retinasbetween diabetic and normal rats while no obvious morphological changes in retinas of 8 weeks diabetic rats could be found, whichindicated that protein expressional changes precededhistological changesin retinae of diabetic rat.These proteins, such as crystallin alpha B, Albumin, DDAHⅡ, TIM-1,GK-1, Prdx 6 and ATP synthase subunit d, play important roles incytoprotective action, anti-apoptotic functions, nitric oxide signaling, antioxidantactivity, glycolysis, Photoexcitation, ATP synthesis, oxidative stress, and so on.These proteins may involve in the pathogenetic mechanisms andprognosis of diabetic retinopathy, and may be valuable in its diagnosisand therapy.