SRM Research Institute and Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, 603 203 Tamilnadu, India.
Department of Life Sciences, Central University of Tamil Nadu, Tiruvarur 610005, India.
Oxid Med Cell Longev. 2021 Apr 8;2021:2732435. doi: 10.1155/2021/2732435. eCollection 2021.
Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases.
内质网 (ER) 在蛋白质的折叠、修饰和运输中发挥着关键作用。当 ER 的内稳态受到干扰时，未折叠或错误折叠的蛋白质在 ER 中积累，导致 ER 应激。持续的 ER 应激导致细胞凋亡，这与各种疾病有关。核因子红细胞 2 相关因子 2 (Nrf2) 是一种主要的转录因子，通过调节与解毒和细胞保护机制相关的各种基因来调节氧化还原平衡。我们发现，用酶片段互补测定法，丹酚酸 A (RA) 处理剂量依赖性地激活内皮细胞中的 Nrf2。本研究探讨了 RA 对内质网应激诱导的内皮细胞凋亡的保护作用及其分子机制。用 qPCR 测定内质网应激暴露的内皮细胞中 Nrf2 及其靶基因和内质网应激标志物的表达。用 Nrf2-CRISPR/Cas9 敲除研究建立了 Nrf2 在 RA 介导的内皮细胞防御机制中的作用。用 RA 处理内质网应激诱导的内皮细胞，显示 Nrf2 被激活，Nrf2 易位和内质网应激标志物减少。我们发现，Nrf2 敲除使内皮细胞对内质网应激敏感，进一步表明 RA 无法介导其细胞保护作用。用 LC-MS/MS 进行的蛋白质组学研究表明，在检测到的 1370 种蛋白质中，我们在内质网应激诱导的内皮细胞中发现了 296 种差异调节的蛋白质，RA 给药使 71 种蛋白质的表达水平恢复到对照水平。值得注意的是，内皮细胞中的内质网应激被 RA 的处理所减弱，这表明 Nrf2 激活剂在与内质网应激相关的疾病的病理条件下发挥作用。
