8-hydroxy-2--deoxyguanosine and Uveitis

Purpose. Previous studies indicate that the upregulation of alphaA crystallin prevents photoreceptor mitochondrial oxidative stress-mediated apoptosis in experimental autoimmune uveitis (EAU). In this study, the role of TLR4 was investigated in the upregulation of alphaA crystallin in the retinas of animals with EAU. Methods. TLR4(-/-), iNOS(-/-), TNF-alpha(-/-), MyD88(-/-), wild-type (WT) control (C57BL/6), and nude mice (B6.Cg-Foxn1(nu)) were immunized with IRBP mixed with complete Freund's adjuvant; eyes were enucleated on day 7 after immunization. Real-time polymerase chain reaction was first used to detect upregulated inflammatory cytokines and alphaA crystallin in retinas with EAU; confirmed with Western blot analysis, and the site of upregulation was localized by immunohistochemistry. Oxidative stress was localized using 8-OHdG, and TUNEL staining was used to detect apoptosis. Results. In early EAU, increased expression of TNF-alpha, iNOS, and alphaA crystallin genes were detected in the retinas of WT mice, whereas such upregulation was absent in TLR4-deficient mice (P < 0.001). alphaA Crystallin was not elevated in MyD88(-/-), TNF-alpha(-/-), and iNOS(-/-) mice with EAU. Immunostaining revealed TNF-alpha, iNOS, and alphaA crystallin localization in the photoreceptor inner segments and outer plexiform layer in the WT controls with EAU; but such staining was absent in TLR4-deficient mice with EAU. 8-OHdG staining showed oxidative stress in the photoreceptors in WT mice with EAU and there was no apoptosis. Conclusions. TLR4 plays an important role in the upregulation of alphaA crystallin through the interaction of MyD88 and the subsequent generation of TNF-alpha and iNOS in the EAU retina. Such crystallin upregulation may prevent oxidative stress-mediated apoptosis of photoreceptors in uveitis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; alpha-Crystallin A Chain; Animals; Apoptosis; Autoimmune Diseases; Blotting, Western; Deoxyguanosine; Disease Models, Animal; Eye Proteins; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; NF-kappa B; Nitric Oxide Synthase Type II; Oxidative Stress; Photoreceptor Cells, Vertebrate; Retinol-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Up-Regulation; Uveitis

Resveratrol is known as one of the antioxidant polyphenols contained in red wine and grape skin. The purpose of the present study was to investigate the role of resveratrol in ocular inflammation in endotoxin-induced uveitis (EIU).. EIU was induced in male C57/B6 mice at the age of 6 weeks by a single intraperitoneal injection of lipopolysaccharide (LPS). Animals had received oral supplementation of resveratrol at the doses of 5, 50, 100, or 200 mg/kg for 5 days until LPS injection. Twenty-four hours after LPS administration, leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. Retinal and retinal pigment epithelium (RPE)-choroidal levels of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear translocation of nuclear factor (NF)-kappaB p65 were evaluated by enzyme-linked immunosorbent assay. Retinal and RPE-choroidal activities of silent information regulator two ortholog (SIRT) 1 were measured by deacetylase fluorometric assay.. Resveratrol pretreatment led to significant and dose-dependent suppression of leukocyte adhesion to retinal vessels of EIU mice compared with vehicle application. Protein levels of MCP-1 and ICAM-1 in the retina and the RPE-choroid of EIU animals were significantly reduced by resveratrol administration. Importantly, resveratrol-treated animals showed significant decline of retinal 8-OHdG generation and nuclear NF-kappaB P65 translocation, both of which were upregulated after EIU induction. RPE-choroidal SIRT1 activity, reduced in EIU animals, was significantly augmented by treatment with resveratrol.. Resveratrol prevented EIU-associated cellular and molecular inflammatory responses by inhibiting oxidative damage and redox-sensitive NF-kappaB activation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Oral; Animals; Antioxidants; Blotting, Western; Chemokine CCL2; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Fluorescent Antibody Technique, Indirect; Inflammation; Injections, Intraperitoneal; Intercellular Adhesion Molecule-1; Leukocytes; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; Resveratrol; Retina; Retinal Pigment Epithelium; Retinal Vessels; Sirtuin 1; Sirtuins; Stilbenes; Transcription Factor RelA; Uveitis