Yang Shui-Ping, Yang Xiu-Zhen, Cao Guo-Ping
Department of Ophthalmology, Taizhou People's Hospital, Taizhou, Jiangsu 225499, P.R. China.
Department of Hepatic Disease, Taizhou People's Hospital, Taizhou, Jiangsu 225499, P.R. China.
Mol Med Rep. 2015 Jul;12(1):1145-50. doi: 10.3892/mmr.2015.3490. Epub 2015 Mar 13.
Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment with ALCAR led to significant protection from these effects. The present results suggested that ALCAR either prevents or ameliorates the actions of the antioxidant system in HLECs at the level of the protein and the gene. Further advanced studies are required for the development of ALCAR as an anti-cataract agent.
先前的研究表明，血清同型半胱氨酸（Hcy）水平升高与青少年白内障和年龄相关性白内障的发生密切相关。Hcy浓度升高可能会诱导DNA启动子区域的基因特异性去甲基化。本研究的目的是通过向人晶状体上皮细胞（HLECs）施用乙酰左旋肉碱（ALCAR）来防止这种去甲基化。使用不同浓度的Hcy处理HLECs 3、6、12和24小时，并将结果用于确定诱导内质网（ER）应激的最佳剂量。同样，对ALCAR的浓度进行了标准化。测量了活性氧（ROS）的产生和经历细胞死亡的细胞百分比。还评估了抗氧化剂水平、ER应激相关蛋白、核因子红细胞2相关因子2（Nrf2）、Kelch样ECH相关蛋白1（Keap1）的mRNA水平以及Keap1基因的启动子DNA甲基化。观察到Hcy会诱导ER应激、产生活性氧并导致细胞死亡。然而，施用ALCAR在很大程度上预防了这些效应。此外，蛋白质印迹分析显示，ALCAR增加了抗氧化蛋白的水平，包括过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、Nrf2、Keap1和谷胱甘肽。同样，对Nrf2和Keap1进行的逆转录定量聚合酶链反应实验以及亚硫酸氢盐基因组DNA测序分析显示，ALCAR对Hcy诱导的ER应激具有预防作用。ER应激诱导的未折叠蛋白反应激活负责Keap1启动子DNA的去甲基化，从而激活Keap1蛋白的表达，进而增加Nrf2被蛋白酶体降解的靶向性。Nrf2活性的这种降低会抑制许多抗氧化酶基因的转录，并使氧化还原平衡朝着晶状体氧化的方向改变。然而，用ALCAR治疗可显著预防这些效应。目前的结果表明，ALCAR在蛋白质和基因水平上可预防或改善HLECs中抗氧化系统的作用。将ALCAR开发为抗白内障药物还需要进一步深入的研究。
