Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, China.
Diabetologia. 2011 Feb;54(2):459-68. doi: 10.1007/s00125-010-1943-1. Epub 2010 Oct 27.
AIMS/HYPOTHESIS: Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.
Cultured retinal pigment epithelial cells and retinal capillary endothelial cells were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE), and an antioxidant, N-acetyl-cysteine (NAC). In vivo, rats with streptozotocin-induced diabetes were treated by NAC for 8 weeks. Activation of the canonical WNT pathway was measured by TOPFLASH assay and by western blot analysis of WNT pathway components and a WNT target gene, Ctgf. Oxidative stress in the retina was evaluated by immunostaining of HNE and 3-nitrotyrosine.
Levels of phosphorylated and total LDL receptor-related protein (LRP)6, and cytosolic β-catenin, as well as transcriptional activity of T cell factor (TCF)/β-catenin were significantly increased by HNE. The production of connective tissue growth factor (CTGF) was also upregulated by HNE. NAC blocked the WNT pathway activation induced by HNE. Furthermore, LRP6 stability was increased by HNE and decreased by NAC. Retinal levels of HNE and 3-nitrotyrosine were significantly increased in diabetic rats, compared with those in non-diabetic rats. In the same diabetic rat retinas, levels of LRP6, cytosolic β-catenin and CTGF were significantly increased. NAC treatment reduced HNE and 3-nitrotyrosine levels and attenuated the upregulation of LRP6, β-catenin and CTGF in diabetic rat retina.
CONCLUSIONS/INTERPRETATION: Lipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.
目的/假说：我们最近的研究表明，无翅型 MMV 整合位点（WNT）途径的激活在糖尿病性视网膜病变和年龄相关性黄斑变性中起致病作用。在这里，我们研究了氧化应激在实验性糖尿病模型中视网膜 WNT 途径激活中的因果作用。
用脂质过氧化产物 4-羟基壬烯醛（HNE）和抗氧化剂 N-乙酰半胱氨酸（NAC）处理培养的视网膜色素上皮细胞和视网膜毛细血管内皮细胞。在体内，用 NAC 治疗链脲佐菌素诱导的糖尿病大鼠 8 周。通过 TOPFLASH 测定和 WNT 途径成分和 WNT 靶基因 Ctgf 的 Western blot 分析来测量经典 WNT 途径的激活。通过 HNE 和 3-硝基酪氨酸的免疫染色评估视网膜中的氧化应激。
HNE 显著增加了磷酸化和总 LDL 受体相关蛋白（LRP）6 以及细胞质β-连环蛋白的水平，以及 T 细胞因子（TCF）/β-连环蛋白的转录活性。HNE 还上调了结缔组织生长因子（CTGF）的产生。NAC 阻断了 HNE 诱导的 WNT 途径激活。此外，HNE 增加了 LRP6 的稳定性，而 NAC 则降低了 LRP6 的稳定性。与非糖尿病大鼠相比，糖尿病大鼠视网膜中的 HNE 和 3-硝基酪氨酸水平明显升高。在相同的糖尿病大鼠视网膜中，LRP6、细胞质β-连环蛋白和 CTGF 的水平明显升高。NAC 治疗降低了糖尿病大鼠视网膜中 HNE 和 3-硝基酪氨酸的水平，并减轻了 LRP6、β-连环蛋白和 CTGF 的上调。
结论/解释：脂质过氧化产物通过氧化应激激活经典 WNT 途径，在视网膜疾病的发展中起重要作用。
