The Effect Of 2-SeCD On Conduction Of AGEs Induced In Diabetic Retinopathy

Diabetic retinopathy (DR) is a leading cause of blindness in theworking-age population of most developed countries. Advanced glycation end product is one of pathogenesies of diabetic capillary complications. It plays an important role in the process of diabetic complication, but the exact signal path is not clear. In that case, it is important to approach the possible signal path and to block the path. AGEs is the oxidizing reaction and structural rearrangement end products of protein, lipid, acid or nucleic with glucose. The concentration of the AGEs in the normal adults'blood will increase following the age. AGEs may make oxidative injury to the cells through receptor dependent or non-receptor dependent pathway. It may have the reaction with the NADPH oxidase and the reactive oxygen species. And ROS active the nuclear factor kappa B and phosphokinase C, then make the target gene transcription.2-SeCD is the simulant of glutathione peroxidase which is composed by polymer chemistry lab in Jilin University. The vigor of 2-SeCD is 7.4 U/μmol, and it has a better stability, water solubility. Its substance is carbohydrate, and there is no aromatic ring in its formation. It has potential to be an important antioxygen medicine. The main mechanism of action is to clear the ROS which is the product of the biological oxidation in vivo. The pathogenesies of diabetic capillary complication include: polyalcohol pathway, AGEs, aninohexose pathway and PKC. Some scholar pointed out that the ROS accumulation may be the co-pathogenesy of diabetic capillary complication. So clearance of ROS can be a possible and important target to block the development of diabetic capillary complication.We approach the RPE cells and vascular endothelial cells proliferation activity, PKC pathway and NF-κB affected by AGEs. We use GPX stimulant 2-SeCD to interfere the process of AGEs affect on RPE cells and RF/6A in vitro. We injected the AGEs which is prepared in vitro into the Wistar rats from the tail veins. To directly observe the injury by AGEs to the retina in vivo and to deplete the effect of carbohydrate and ROS the product of other pathway. We also approach the protection by 2-SeCD to the injury induced by AGEs in retina. We hope that 2-SeCD can be a new medicamentum to DR.We cultured the RPE cells and macaque retinal vascular endothelial cell in vitro. The RPE cells is cultured in 10% DMEM low carbohydrates medium. We utilized different concentration 2-SeCD to effect the RPE cells induced by AGEs. The groups divided in RPE cells induced by AGEs are:normal control; low concentration BSA control (50.0 mg·L-1); middle concentration BSA control (100.0 mg·L-1); high concentration BSA control (200.0 mg·L-1); low concentration AGE (50.0 mg·L-1); middle concentration AGE(100. 0mg·L-1); high concentration AGE (200.0mg·L-1). The groups divided in RPE cells effected by 2-SeCD:normal control; BSA control (200.0 mg·L-1); AGE (200.0 mg·L-1); aminoguanidine(AG 320μmol/L+ AGEs 200.0 mg·L-1); 2-SeCD low concentration (2-SeCD 80μmol/L+AGEs200.0 mg·L-1); 2-SeCD mid-concentration (2-SeCD 160μmol/L+AGEs200.0 mg·L-1); 2-SeCD high concentration (2-SeCD 320μmol/L+AGEs200.0 mg·L-1). Then we detected the cells proliferate activation by MTT assay; detected VEGF protein expression by immunochemistry; detected the activation of NF-kappa B by immunofluorescence; detected the concentration of MDA in medium of RPE cells; detected the VEGF and PKCβⅡprotein level by western blot. The experiments results:(1) the VEGF detected by immunochemistry, the VEGF shows masculine expression in all groups, it shows the yellow and buffy grains in kytoplasm. The color of the grains darkens in accompany of the increase concentration. (2) the immunofluorescence shows that NF-κB p65 masculine expression transfer into nucleus in AGE groups, but it has no concentration dependent. (3) the western blot shows that the VEGF protein level step up obviously in low, middle, high concentration AGEs groups contrast to normal control group. The VEGF protein level in high concentration AGEs group is higher obviously than the low and middle AGEs group. (3) The MTT assay shows the cells proliferation activation:RPE cells proliferation activation decrease in the AGEs (200.0 mg·L-1)group; RPE cells proliferation increase obviously in the 2-SeCD high concentration group than the AGEs (200.0 mg·L-1)group. (4) To detect MDA in the cell medium:the MDA concentration in the cell medium in 2-SeCD high concentration group is higher obviously than the AGEs (200.0 mg·L-1)group. (5) Western Blot detected the PKCβⅡprotein level:the PKCβⅡprotein level is lower in the high concentration 2-SeCD group than the low, middle concentration 2-SeCD group, AGEs group and AG group.We also cultured the RF/6A cells in 10% RPMI-1640 medium. We detected the cells proliferate activation by MTT assay; detected NOX2, NOX3, NF-kappa B and VEGF protein expression by immunochemistry; detected the concentration of MDA in medium of RPE cells; detected the VEGF m RNA level by RT-PCR; detect the PKCβⅡprotein level by western blot. The experiments results:(1) MTT assay:the AGEs can suppress the RF/6A cells proliferation. The RF/6A cells proliferation increased in the high concentration 2-SeCD group compared with AGEs groups. (2) Immunochemistry:The NOX2, NF-κB p65 and VEGF protein expression decreased in the high concentration 2-SeCD group than AGEs groups. (3) RT-PCR:The VEGF m RNA level in high concentration 2-SeCD group is higher than AGEs groups. (4) Western Blot:The PKCβⅡprotein level in the high concentration 2-SeCD group is higher than AGEs groups.We divided 50 Wistar rats into five groups:normal control group (CON,n=10); RSA control group (RSA, n=10); AGEs group (AGE, n=10) aminoguanidine group (AGE+AG, n=10); 2-SeCD group (AGE+CD, n=10) There is no treatment to the CON group; to inject RSA 100 mg/kg each day from tail vein to RSA group; to inject AGEs 100 mg/kg each day to vena caudalis to AGE group; to inject intraperitoneal aminoguanidine 40 mg/kg, then 30 minutes later inject AGE 100 mg/kg to vena caudalis each day in AG group; to inject intraperitoneal 2-SeCD 16μmol/kg, then 30 minutes later inject AGE 100 mg/kg to vena caudalis each day in AG group. We take fluorescein fundus angiography to the rats one month later to observe the retinal vessels; detect the blood-serum MDA concentration and blood sugar level; take electron microscope to the retina, HE drum dyeing; immunohistochemistry to detect the retina NF-κB,VEGF protein expression; to detect the retina VEGF,PDGF mRNA level by RT-PCR. The results:(1) FFA shows 6 to 8 retinal artery and veins diverge from optic disc. There is no avascular area or macula lutea-like structure in CON group. The terminations of the retinal capillary expand, the vasopermeability increase, and there is fluorescein leakage around the termination of the retinal capillary. The retinal veins dilat and distort in AGE group. The RSA group shows the same as CON group. The FFA in AG group and 2-SeCD group show veins dilat and distort, but no fluorescein leakage. (2) MDA concentration:the AGE group is higher obviously than CON, RSA and CD groups. The blood-serum MDA concentration in CD group is lower than the AG group.The conclusions are:AGEs may cause the ROS cumulation through the NADPH oxidase pathway, then activate the PKC and NF-κB signal trans-path and the VEGF up-regulation. In our research we use the GPX stimulant which is composed in our cooperational lab to interfere the AGEs damage. It points out a new way and a new medicine for the prevention and cure to the DR in earlier period.