Department of Ophthalmology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan , China.
Department of Ophthalmology, People's Hospital of Deyang City, Deyang, Sichuan, China.
Transl Vis Sci Technol. 2024 Mar 1;13(3):19. doi: 10.1167/tvst.13.3.19.
The regulation of mitophagy by Sirt3 has rarely been studied in ocular diseases. In the present study, we determined the effects of Sirt3 on AMPK/mTOR/ULK1 signaling pathway-mediated mitophagy in retinal pigment epithelial (RPE) cells in a high glucose environment.
The mRNA expression levels of Sirt3, AMPK, mTOR, ULK1, and LC3B in RPE cells under varying glucose conditions were measured by real-time polymerase chain reaction (RT-PCR). The expressions of Sirt3, mitophagy protein, and AMPK/mTOR/ULK1 signaling pathway-related proteins were detected by Western blotting. Lentivirus (LV) transfection mediated the stable overexpression of Sirt3 in cell lines. The experimental groups were NG (5.5 mM glucose), hypertonic, HG (30 mM glucose), HG + LV-GFP, and HG + LV-Sirt3. Western blotting was performed to detect the expressions of mitophagy proteins and AMPK/mTOR/ULK1-related proteins in a high glucose environment during the overexpression of Sirt3. Reactive oxygen species (ROS) production in a high glucose environment was measured by DCFH-DA staining. Mitophagy was detected by labeling mitochondria and lysosomes with MitoTracker and LysoTracker probes, respectively. Apoptosis was detected by flow cytometry.
Sirt3 expression was reduced in the high glucose group, inhibiting the AMPK/mTOR/ULK1 pathway, with diminished mitophagy and increased intracellular ROS production. The overexpression of Sirt3, increased expression of p-AMPK/AMPK and p-ULK1/ULK1, and decreased expression of p-mTOR/mTOR inhibited cell apoptosis and enhanced mitophagy.
Sirt3 protected RPE cells from high glucose-induced injury by activating the AMPK/mTOR/ULK1 signaling pathway.
By identifying new targets of action, we aimed to establish effective therapeutic targets for diabetic retinopathy treatment.
Sirt3 对眼部疾病中线粒体自噬的调节作用研究甚少。本研究旨在探讨 Sirt3 对高糖环境下视网膜色素上皮（RPE）细胞 AMPK/mTOR/ULK1 信号通路介导的线粒体自噬的影响。
采用实时聚合酶链反应（RT-PCR）检测不同葡萄糖条件下 RPE 细胞中 Sirt3、AMPK、mTOR、ULK1 和 LC3B 的 mRNA 表达水平。采用 Western blot 检测 Sirt3、线粒体自噬蛋白及 AMPK/mTOR/ULK1 信号通路相关蛋白的表达。通过慢病毒（LV）转染实现 Sirt3 在细胞系中的稳定过表达。实验分组为 NG（5.5mmol/L 葡萄糖）、高渗、HG（30mmol/L 葡萄糖）、HG+LV-GFP 和 HG+LV-Sirt3。在高糖环境中过表达 Sirt3 后，通过 Western blot 检测线粒体自噬蛋白和 AMPK/mTOR/ULK1 相关蛋白的表达。采用 DCFH-DA 染色检测高糖环境下活性氧（ROS）的产生。分别用 MitoTracker 和 LysoTracker 探针标记线粒体和溶酶体来检测线粒体自噬。采用流式细胞术检测细胞凋亡。
高糖组 Sirt3 表达下调，抑制 AMPK/mTOR/ULK1 通路，导致线粒体自噬减少，细胞内 ROS 生成增加。Sirt3 过表达增加 p-AMPK/AMPK 和 p-ULK1/ULK1 的表达，降低 p-mTOR/mTOR 的表达，抑制细胞凋亡，增强线粒体自噬。
Sirt3 通过激活 AMPK/mTOR/ULK1 信号通路，保护 RPE 细胞免受高糖诱导的损伤。
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