Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada.
Mudanjiang Medical University, Heilongjiang, PR China.
Physiol Rep. 2020 Feb;8(3):e14331. doi: 10.14814/phy2.14331.
Diabetic complications cause significant morbidity and mortality. Dysfunction of vascular endothelial cells (ECs), caused by oxidative stress, is a main mechanism of cellular damage. Oxidative stress accelerates EC senescence and DNA damage. In this study, we examined the role of mitochondrial sirtuins (SIRTs) in glucose-induced oxidative stress, EC senescence, and their regulation by miRNAs. Human retinal microvascular endothelial cells (HRECs) were exposed to 5 mmol/L (normoglycemia; NG) or 25 mmol/L glucose (hyperglycemia; HG) with or without transfection of miRNA antagomirs (miRNA-1, miRNA-19b, and miRNA-320; specific SIRT-targeting miRNAs). Expressions of SIRT3, 4 and 5 and their targeting miRNAs were examined using qRT-PCR and ELISAs were used to study SIRT proteins. Cellular senescence was investigated using senescence-associated β-gal stain; while, oxidative stress and mitochondrial alterations were examined using 8-OHdG staining and cytochrome B expressions, respectively. A streptozotocin-induced diabetic mouse model was also used and animal retinas and hearts were collected at 2 months of diabetes. In HRECs, HG downregulated the mRNAs of SIRTs, while SIRT-targeting miRNAs were upregulated. ELISA analyses confirmed such downregulation of SIRTs at the protein level. HG additionally caused early senescence, endothelial-to-mesenchymal transition and oxidative DNA damage in ECs. These changes were prevented by the transfection of specific miRNA antagomirs and by resveratrol. Retinal and cardiac tissues from diabetic mice also showed similar reductions of mitochondrial SIRTs. Collectively, these findings demonstrate a novel mechanism in which mitochondrial SIRTs regulate glucose-induced cellular aging through oxidative stress and how these SIRTs are regulated by specific miRNAs. Identifying such mechanisms may lead to the discovery of novel treatments for diabetic complications.
糖尿病并发症导致显著的发病率和死亡率。氧化应激引起的血管内皮细胞（EC）功能障碍是细胞损伤的主要机制。氧化应激加速 EC 衰老和 DNA 损伤。在这项研究中，我们研究了线粒体沉默调节蛋白（SIRTs）在葡萄糖诱导的氧化应激、EC 衰老中的作用及其受 miRNAs 的调节。将人视网膜微血管内皮细胞（HRECs）暴露于 5 mmol/L（正常血糖；NG）或 25 mmol/L 葡萄糖（高血糖；HG）中，同时转染 miRNA 反义寡核苷酸（miRNA-1、miRNA-19b 和 miRNA-320；特定的 SIRT 靶向 miRNA）。使用 qRT-PCR 检测 SIRT3、4 和 5 的表达及其靶向 miRNA，使用 ELISA 研究 SIRT 蛋白。使用衰老相关β-半乳糖苷染色研究细胞衰老；而使用 8-OHdG 染色和细胞色素 B 表达研究氧化应激和线粒体改变。还使用链脲佐菌素诱导的糖尿病小鼠模型，在糖尿病 2 个月时收集动物的视网膜和心脏。在 HRECs 中，HG 下调 SIRTs 的 mRNA，而 SIRT 靶向 miRNA 上调。ELISA 分析证实了 SIRTs 在蛋白质水平上的下调。HG 还导致 EC 早期衰老、内皮-间质转化和氧化 DNA 损伤。这些变化可以通过转染特定的 miRNA 反义寡核苷酸和白藜芦醇来预防。糖尿病小鼠的视网膜和心脏组织也显示出类似的线粒体 SIRTs 减少。总之，这些发现表明了一种新的机制，即线粒体 SIRTs 通过氧化应激调节葡萄糖诱导的细胞衰老，以及这些 SIRTs 如何受特定 miRNA 的调节。确定这些机制可能会发现治疗糖尿病并发症的新方法。
