Purpose

Increased permeability of retinal capillary endothelial cells is a key feature in the progression of diabetic retinopathy (DR). Precisely why and how diabetes causes dysfunction in retinal capillary endothelial cells is not well understood, making it challenging to explore more advanced therapeutics.

Methods

Cell proliferation was assessed by the Cell Counting Kit-8 assay. Ferroptosis was evaluated by measuring lipid reactive oxygen species levels by flow cytometry and determining malondialdehyde, superoxide dismutase, and glutathione peroxidase levels through biochemical assays. Western blot analysis and quantitative PCR were respectively used to check the expression of proteins and RNAs. Co-immunoprecipitation assays were used to confirm the interaction between TRIM46 and GPX4.

Results

High glucose (HG, 25 mM glucose) significantly suppressed cell growth, which could be reversed by the ferroptosis inhibitor, ferrostatin-1. HG treatment time-dependently induced ferroptosis in human retinal capillary endothelial cells (HRCECs) and induced TRIM46 expression. Lentiviral-mediated overexpression of TRIM46 decreased cell resistance against HG-induced ferroptosis, whereas knockdown showed the opposite effect. Administration of RSL3, a ferroptosis agonist, was able to reverse the protective effects of TRIM46 silencing. TRIM46 interacted with GPX4, an important enzyme that suppresses ferroptosis, and promoted GPX4 ubiquitination. Furthermore, lentiviral-mediated overexpression ofGPX4 ameliorated the effects of TRIM46 overexpression and conferred protection to cells against HG-induced ferroptosis.

Conclusion

TRIM46 and GPX4 form a regulatory pathway that controls HG-induced ferroptosis of HRCECs. Inhibiting this pathway or sustaining the expression of GPX4 enables cells to resist damage caused by HG. We provide new mechanistic insight into the pathology of DR and identified TRIM46 and GPX4 as two molecular targets for the development of effective drugs for DR treatment.

中文翻译：

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TRIM46通过促进GPX4泛素化促进高葡萄糖诱导的人视网膜毛细血管内皮细胞的铁死亡和细胞生长抑制

目的

视网膜毛细血管内皮细胞通透性增加是糖尿病视网膜病变 (DR) 进展的关键特征。糖尿病导致视网膜毛细血管内皮细胞功能障碍的确切原因和方式尚不清楚，这使得探索更先进的治疗方法具有挑战性。

方法

通过 Cell Counting Kit-8 分析评估细胞增殖。通过流式细胞术测量脂质活性氧水平并通过生化分析确定丙二醛、超氧化物歧化酶和谷胱甘肽过氧化物酶水平来评估铁死亡。分别使用蛋白质印迹分析和定量PCR来检查蛋白质和RNA的表达。共免疫沉淀试验用于确认 TRIM46 和 GPX4 之间的相互作用。

结果

高葡萄糖（HG，25 mM 葡萄糖）显着抑制细胞生长，这可以被铁死亡抑制剂 ferrostatin-1 逆转。HG 治疗时间依赖性地诱导人视网膜毛细血管内皮细胞 (HRCEC) 中的铁死亡并诱导 TRIM46 表达。慢病毒介导的 TRIM46 过表达降低了细胞对 HG 诱导的铁死亡的抵抗力，而敲低显示出相反的效果。RSL3 是一种铁死亡激动剂，能够逆转 TRIM46 沉默的保护作用。TRIM46 与 GPX4（一种抑制铁死亡的重要酶）相互作用并促进 GPX4 泛素化。此外，慢病毒介导的 GPX4 过表达改善了 TRIM46 过表达的影响，并为细胞提供了针对 HG 诱导的铁死亡的保护。

结论

TRIM46 和 GPX4 形成调控 HG 诱导的 HRCECs ferroptosis 的调控途径。抑制该通路或维持 GPX4 的表达使细胞能够抵抗 HG 造成的损伤。我们提供了对 DR 病理学的新机制见解，并确定了 TRIM46 和 GPX4 作为开发有效药物治疗 DR 的两个分子靶点。