Wu Pei-Yuan, Ji Ning, Wu Chong-Guang, Wang Xiao-Die, Liu Xin, Song Zhi-Xue, Khan Murad, Shah Suleman, Du Ying-Hua, Wang Xiu-Fang, Yan Li-Fang
Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang 050017, Hebei Province, China.
Department of Ophthalmology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China.
Int J Ophthalmol. 2023 Feb 18;16(2):178-190. doi: 10.18240/ijo.2023.02.03. eCollection 2023.
To determine whether an antisense RNA corresponding to the human Alu transposable element (Aluas RNA) can protect human lens epithelial cells (HLECs) from methylglyoxal-induced apoptosis.
Cell counting kit-8 (CCK-8) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess HLEC viability. HLEC viability/death was detected using a Calcein-AM/PI double staining kit; the annexin V-FITC method was used to detect HLEC apoptosis. The cytosolic reactive oxygen species (ROS) levels in HLECs were determined using a reactive species assay kit. The levels of malondialdehyde (MDA) and the antioxidant activities of total-superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were assessed in HLECs using their respective kits. RT-qPCR and Western blotting were used to measure mRNA and protein expression levels of the genes.
Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxal-induced decrease in Bcl-2 mRNA and the methylglyoxal-induced increase in Bax mRNA. In addition, Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression. Aluas RNA inhibited the production of ROS induced by methylglyoxal, restored T-SOD and GSH-Px activity, and moderated the increase in MDA content after treatment with methylglyoxal. Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes, including glutathione peroxidase, heme oxygenase 1, γ-glutamylcysteine synthetase and quinone oxidoreductase 1. Aluas RNA ameliorated methylglyoxal-induced increases of the mRNA and protein expression of Keap1 that is the negative regulator of Nrf2.
Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.
确定与人类Alu转座元件对应的反义RNA（Aluas RNA）是否能保护人晶状体上皮细胞（HLECs）免受甲基乙二醛诱导的凋亡。
使用细胞计数试剂盒-8（CCK-8）和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法评估HLEC活力。使用钙黄绿素-AM/PI双染试剂盒检测HLEC活力/死亡情况；采用膜联蛋白V-FITC法检测HLEC凋亡。使用活性氧检测试剂盒测定HLECs胞质活性氧（ROS）水平。使用各自的试剂盒评估HLECs中丙二醛（MDA）水平以及总超氧化物歧化酶（T-SOD）和谷胱甘肽过氧化物酶（GSH-Px）的抗氧化活性。采用RT-qPCR和蛋白质印迹法测量基因的mRNA和蛋白质表达水平。
Aluas RNA挽救了甲基乙二醛诱导的HLECs凋亡，并改善了甲基乙二醛诱导的Bcl-2 mRNA降低以及甲基乙二醛诱导的Bax mRNA升高。此外，Aluas RNA抑制了甲基乙二醛诱导的Alu正义RNA表达增加。Aluas RNA抑制了甲基乙二醛诱导的ROS产生，恢复了T-SOD和GSH-Px活性，并减轻了甲基乙二醛处理后MDA含量的增加。Aluas RNA显著恢复了甲基乙二醛诱导的Nrf2基因和抗氧化防御基因（包括谷胱甘肽过氧化物酶、血红素加氧酶1、γ-谷氨酰半胱氨酸合成酶和醌氧化还原酶1）的下调。Aluas RNA改善了甲基乙二醛诱导的Nrf2负调节因子Keap1的mRNA和蛋白质表达增加。
Aluas RNA通过增强抗氧化防御减少甲基乙二醛诱导的凋亡。
