Age-related macular degeneration is a progressive ocular disease that is the leading cause of vision loss among elderly. AMD usually is divided into two types: wet and dry AMD, which is linked with inflammation. Choroidal Neovascularization (CNV) formation or wet AMD is also associated with oxidative stress. Previously, TSP1 has been shown to have a significant alleviating effect on CNV in TSP1 knockout (TSP1-/-) mice. However, the mechanism by which TSP1 ameliorates CNV remains unclear. Here we report that TSP1 reduces nitric oxide production to prevent cells from forming tubes formation and reduced the levels of vascular endothelial growth factor (VEGF) and lipid peroxides (LPO) during oxidative stress. We measured RF/6A cell viability by CCK-8 assay and apoptosis by flow cytometry. RF/6A cell were transfected with TSP1 and STAT3 overexpression, and then the mRNA and protein levels of TSP1 and also the signal pathways were detected by qRT-PCR and Western blot analysis. Migration assays were performed using a transwell system. Co-Immunoprecipitation was used to analyze the binding relationship between CD47 and SHP-2. The results show that overexpression of TSP1 alleviated the damage of oxidative stress to RF/6A cells including increased cell activity and migration, decreased apoptosis and reduced migration compared to the control group. SHP-2 was activated by TSP1 through its receptor CD47 and STAT3 phosphorylation was reduced by activation of SHP-2, thereby blocking STAT3-iNOS pathway and reducing NO concentration in RF/6A cells ultimately protecting them from oxidative stress. Finally, the CNV mice model confirmed that TSP1 overexpression could protect the mice against CNV in vivo, modified the antioxidants levels and decreased the expression of TNF-α and IL-6 under laser irradiation. These results indicate a potential mechanism of TSP1 to slow down formation of CNV in wet AMD, which may bring hope for new treatment strategies.

中文翻译：

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TSP1通过调节STAT3-iNOS信号通路改善与年龄有关的黄斑变性。

与年龄有关的黄斑变性是一种进行性眼病，是老年人视力丧失的主要原因。AMD通常分为两种类型：湿性AMD和干性AMD，这与炎症有关。脉络膜新生血管形成（CNV）或湿性AMD也与氧化应激相关。以前，已证明TSP1对TSP1基因敲除（TSP1-/-）小鼠的CNV有明显的缓解作用。但是，TSP1改善CNV的机制仍不清楚。在这里，我们报告TSP1减少一氧化氮的产生，以防止细胞形成管形成，并降低了氧化应激期间血管内皮生长因子（VEGF）和脂质过氧化物（LPO）的水平。我们通过CCK-8分析测量了RF / 6A细胞的生存能力，并通过流式细胞术测量了细胞凋亡。用TSP1和STAT3过表达转染RF / 6A细胞，然后通过qRT-PCR和Western blot分析检测TSP1的mRNA和蛋白水平以及信号通路。使用transwell系统进行迁移测定。使用共免疫沉淀法分析CD47与SHP-2之间的结合关系。结果表明，与对照组相比，TSP1的过表达减轻了RF / 6A细胞的氧化应激损伤，包括细胞活性和迁移增加，凋亡减少和迁移减少。TSP1通过其受体CD47激活了SHP-2，并且通过激活SHP-2减少了STAT3磷酸化，从而阻断STAT3-iNOS途径并降低RF / 6A细胞中的NO浓度，从而最终使其免受氧化应激。最后，CNV小鼠模型证实TSP1过表达可以保护小鼠体内免受CNV侵害，改善了抗氧化剂的水平，并降低了激光照射下TNF-α和IL-6的表达。这些结果表明，TSP1可能减缓湿性AMD中CNV的形成，这可能为新的治疗策略带来希望。