Ive pressure. As shown in Figure 7A, FeTPPS significantly suppressed HG-induced LRP6 phosphorylation and b-catenin accumulation in a concentration-dependent manner. Comparable to UA, FeTPPS dose-dependently inhibited Wnt ligand-induced LRP6 phosphorylation and b-catenin accumulation (Fig. 7B). Furthermore, FeTPPS attenuated WCM-induced b-catenin transcriptional activity inside a concentration-dependent manner, as shown by Luciferase assay (Fig. 7C).LIU ET AL.FIG. 5. UA inhibited HGactivated Wnt signaling. ARPE19 cells had been cultured with 30 mM glucose for 48 h then treated with UA for another 16 h. pLRP6 and total LRP6 had been measured by Western blot analysis (A) and semiquantified by densitometry (B). Phosphorylated b-catenin (p-b-catenin) and total b-catenin were measured by Western blot evaluation (C) and semiquantified by densitometry (D). Levels of cytosolic (E) and nuclear bcatenin (G) have been determined by Western blot evaluation in subcellular fractions and semiquantified by densitometry (F, H). Values are expressed as mean S.D. **p 0.01 versus LG, {p 0.05 and { p 0.01 versus HG.Clinical characteristics of diabetic animals To determine the effect of nitrosative stress on Wnt signaling in the retina, we used streptozotocin (STZ)-induced diabetes rats, which exhibit Wnt pathway overactivation in the retina (8). Before the STZ injection, all of these rats had similar blood glucose levels (*100 mg/dl) and body weights (*140 g). Upon onset of STZ-induced diabetes, the diabetic rats were randomly assigned to two groups. One group was fed with UA (160 mg/kg/d) in drinking water for 6 weeks, and the other group was fed with regular drinking water as diabetic control. Six weeks after the treatment, no significant difference was observed in blood glucose levels and body weights between UA-fed diabetic and untreated diabetic control rats (Table 1) at any of the time points, suggesting that oral administration of UA had no effect on hyperglycemia and body weight loss in the diabetic rat model.UA suppresses Wnt signaling in the retina of diabetic rats To investigate whether UA inhibits the Wnt-signaling pathway in vivo, we fed the diabetic rats with UA. As shown by immunoblotting, 3-NT levels were significantly increased in the retinas of diabetic rats, compared to the nondiabetic control (Fig. 8A). Immunostaining using an antibody specific for 3-NT showed more intense 3-NT signals in the diabetic rat retinas, primarily in the inner retina (Fig. 8B). Treatment of diabetic rats with (160 mg/kg/d) UA for 6 weeks markedly reduced 3-NT levels in the retina as indicated by both Western blot analysis and immunostaining (Fig.Fluralaner 8A, B).(S)-(-)-Levamisole Retinal levels of pLRP6, total LRP6, and b-catenin were significantly upregulated in diabetic rat retinas, consistent with our previous study (Fig.PMID:23907051 8C ). Nuclear translocation of b-catenin was also increased in the retina of the diabetic rats (Fig. 8G). UANITROSATIVE STRESS IN DIABETIC RETINOPATHYFIG. 6. UA inhibited the Wnt pathway activation induced by Wnt3A, but not that induced by an active mutant of b-catenin. (A, B) ARPE19 cells were treated with a Wnt3a-conditioned medium (WCM) for 16 h with or without UA. pLRP6 and total LRP6 were measured by Western blot analysis (A) and semiquantified by densitometry (B). (C) Human telomerase reverse transcriptaseimmortalized retinal pigment epithelial (HTERT-RPE) cells were transfected with TOPFLASH or FOPFLASH plasmid for 8 h and then treated with WCM for 16 h with or without UA. (D) The.
