Analysis Of Peroxiredoxin 6 Decreasing Oxidative Stress In Diabetic Retinopathy

AMD is an age-related, progressive degener-ation of photoreceptors and their underlying retinal pigment epithelium (RPE) in the macular area of the retina. Although the vision loss results from photoreceptor damage in the central retina, the initial pathogenesis of AMD involves the degeneration of RPE. Most likely, multiple factors are involved in AMD, but the oxidative stress may play an important role. Similar to other parts of the body, RPE is also subjected to age-related oxidation and the resultant oxidative stress. The lesions induced by oxidative injury may accumulate and trigger a.ected RPE cells to undergo apoptosis. So everyone with different genetic characteristics, oxidative stress may be particularly significant in the RPE, and the available evidence indicates that oxidative stress-induced apoptosis involves a signaling mechanism generated from mitochondria. Direct intake of antioxidants or induction of antioxidants synthesis provides ways to protect the RPE antioxidant state, and delay the development of AMD.Diabetic retinopathy, a disease of the retina, is the leading cause of acquired blindness in adults. Diabetes results in increased oxidative stress, and elevated oxidative stress plays an important role in the pathogenesis of diabetic complications. The retina has high content of polyunsaturated fatty acids and has the highest oxygen uptake and glucose oxidation relative to any other tissue. This phenomenon renders retina more susceptible to oxidative stress. Since oxidative stress represents an imbalance between excess formation and/or impaired removal of ROS, the antioxidant defense system of the cell is a crucial part of the overall oxidative stress experienced by a cell. In diabetes, the activities of antioxidant defense enzymes responsible for scavenging free radicals and maintaining redox homeostasis such as SOD, glutathione reductase, glutathione peroxidase, and catalase are diminished in the retina.So oxidative stress can lead to diabetic retinopathy in viarous ways. It is important that we find ways to treat the disease, retard the development of the disease.Peroxiredoxins (PRDXs) are a newly identified family of non-selenium glutathione peroxidases that have been reported to be present in many major organs, including the lens and the retinal. Substantial evidence suggests that they are involved in balancing the oxidant–antioxidant system by removing or limiting reactive oxygen species (ROS), thereby acting as protector proteins. Most members of Prx family contain two conserved cysteines for catalytic sites and are thus referred to as 2-cysPrx. The 1-cysPrx (peroxiredoxin 6 [Prdx6], antioxidant protein 2 [AOP2]), on the other hand, contains a single conserved cysteine residue that plays the critical role in the active site for the reduction of phospholipid hydroperoxides and other peroxides. Peroxidase activity for what is now called peroxiredoxin 6 (Prdx6) was first demonstrated for protein isolated from the ciliary body of the bovine eye.We are to construct pEGFP-Prx6 eukaryotic expressing vector, and to study the overexpression of Prx6 after transduction with Lipdectamine 2000 increased resistance to oxidative stress of RPE cell and the retinal of the diabetic.Objective:This study is to construct the recombinant eukaryotic expressing vector pEGFP-Prx6 through cloning gene of Prx6, obtain stable clone expression of Prx6 gene and investigate the suppressing effect of Prx6 to RPE injury induced by hydrogen peroxide(H2O2),and transfect the retina of the diabetic rat to study the antioxidant and antiapopsis of Prx6,and to search new target for preventing and curing DR.Methods:1. Cultured human RPE cells were stimulated with H2O2 at different doses (0, 0.125, 0.25, 0.5 and 1 mmol·L-1) for 24 hours. Subsequently, the cells were washed and resuspended in the culture media. The expression of Prx6 was evaluated by immunohistochemistry and RT-PCR, and the levels of Prx6 were compared with levels in control cells. The effect of H2O2 on RPE cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) testing.2. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of PRDX6, rats were killed at 4, 8, and 12 weeks after the injection of STZ, and PRDX6 expression in the retina from both control and STZ-induced diabetic rats was measured by reverse transcription polymerase chain reaction, and immunohistochemistry.3. Prx6 gene segment obtained from human lung cancer by RT-PCR, was sub-cloned into eukaryotic expressing vector pEGFP-N1 twice, then the obtained recombinant eukaryotic expressing plasmid pEGFP-Prx6 was identified with sequence analysis.4. pEGFP-Prx 6 plasmid was constructed and stably transfected into human RPE cells. RT-PCR and immunochemochal analysis was used to monitor expression of Prx6 in the stable transfectants.5. RPEs were divided into three groups: non-transfected group,empty plasmid group and transfected group. All the cells were attacked with H2O2 of different concentration. Their proliferative activity was examined by MTT assay. And the cells were attacked with high glucose of different time. Their proliferative activity was also examined by MTT assay.6. 21 diabetic rats were randomly divided into 3 groups as pEGFP-Prx6 group, liposome group and control group. Lipofectamine 2000 and pEGFP-Prx6 plasmids were intraviteal injected in the eyes for Prx6 group, pure Lipofectamine 2000 were intraviteal injected in eyes for liposome group. The control groups were not interfered. 1d,2w,3w,4w after the injection was performed, RT-PCR,Western Blot,Tunnel were performed to evaluate the effect of Prx6 on experimental diabetic retinopathy.Results:1. There are expression of Prx6 in normal RPE cells when evaluated by immunohistochemistry and RT-PCR. Exposure of RPE cells to 0.125 mmol·L-1 H2O2 showed a significant increase (4-fold) in Prx6, but when exposed to 0.5 and 1 mmol·L-1 H2O2, there was a great decrease in Prx6. The viability of ARPE-19 cells were slightly decreased when the concentrations of H2O2 were 0.125 mmol·L-1(91%), but the cells showed progressive loss of viability at concentrations of 0.25, 0.5 and 1 mmol·L-1 H2O2 (83%,74%,52%).2. The result of immunohistochemistry showed that Prx6 was observed in the outer nuclear layer (ONL) and the inner nuclear layer (INL) in normal rat. A low but significant level of expression was observed in the ganglion cell layer (GCL). Intense Prx6 staining was present in the INL and the ONL in 4 weeks of diabetic rats. The retina of 8 and 12 weeks after STZ administration revealed lack of staining (F=22967.63 P<0.05) . Subsequently, expression of Prx6 mRNA in the retinal of rat was evaluated. The result was similar to that of immunohistochemistry, which resulted in a markedly increased expression of Prx6 mRNA 4 weeks after STZ administration and a markedly decreased expression of Prx6 mRNA 8 and 12 weeks after STZ administration (F=942.84 P<0.05) .3. Electrophoresis proved that total RNA extracted from lung cancer is intact.DNA sequencing make it clear that cDNA sequence of Prx6 is the same as the sequence in Genebank.The sequencing results showed that Prx6 gene segment had been coloned into recombinant eukaryotic expressing vector pEGFP-Prx6.4. The plasmid pEGFP-Prx6 was insert successfully into genome DNA of RPEs by PCR. Prx6 mRNA level in transfected group was higher than in non-transfected group and empty plasmid group(P<0.01).The results of MTT assay showed that H2O2 inhibited the proliferation of cell through a kind of H2O2 concentration-dependent manner. Compared with non-transfected group,empty vector does not interfere with the growth of RPEs(P=0.631), while the transfected Prx6 gene can weaken the inhibition of H2O2 against RPEs(P<0.05).5. The plasmid pEGFP-Prx6 was transfected into RPEs. Prx6 mRNA level in transfected group was higher than in non-transfected group(P<0.01).The results of MTT assay showed that high glucose inhibited the proliferation of cell through a kind of time-dependent manner. Compared with non-transfected group, the transfected Prx6 gene can weaken the inhibition of high glucose against RPEs(P<0.05).6. The plasmid pEGFP-Prx6 was transfected successfully into the diabetic rat eyes. Prx6 mRNA level in transfected group was higher than in empty plasmid group on the first day, and continued for 4 weeks(P<0.01). Expression of Prx6 protein in transfected group was higher than in empty plasmid group with Western Blot analysis(P<0.01). The Tunel analysis shows that overexpression of Prx6 could protect the diabetic rat retina against oxidative stress.Conclusions:1. There are expression of Prx6 in RPE cells. In the attack of H2O2 with different concentrate, the expression of Prx6 decreases in a dose dependent manner.2. There are expression of Prx6 in the retina of rat. With the development of diabetic retinopathy, the expression of Prx6 decreases and disappears at last in a time dependent manner.3. We constructed pEGFP-Prx6 eukaryotic expressing vector and tranfected the RPE cell and the retinal of the diabetic successfully.4. Overexpression of Prx6 in RPE cell can protect the cell against the oxidative stress.5. Overexpression of Prx6 in the retina of diabetic rat can protect the retina against the oxidative stress.