Extremophiles Lab, Department of Microbiology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, 1417466191, Iran.
Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, 1417466191, Iran.
Sci Rep. 2019 Mar 1;9(1):3248. doi: 10.1038/s41598-019-39011-w.
The major signaling pathway in human cells is related to the antioxidant defense system. The main component of this system is a transcription factor, Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). It regulates this system in different cellular situations under stimulation by oxidative stress or antioxidants. Thus, detecting the stimulation of NRF2 via a screening strategy may enable us to discover stimulating agents of NRF2-related signaling pathway. With this in mind, we designed a whole cell bioreporter containing the NRF2 response elements that are inserted in a luciferase vector, immediately upstream of a luciferase gene whose promoter has been removed. This bioreporter is activated by stimulators such as 3H-1,2-dithiole-3-thione (D3T), butyl hydroxyanisole (BHA) and ascorbic acid reacting as antioxidant agents. It was observed that the regulatory region of the NRF2 gene, which is identified by NRF2 protein, is located inside its coding region. This designed bioreporter can detect the presence of antioxidant agents. It also exhibits a significant linear correlation over different doses of these agents ranging from 0.8 to 80 μM for ascorbic acid, 0.1 to 100 μM for D3T, and 0.1 to 100 μM for BHA. This detection system is proven to be more sensitive than Real-time PCR, suggesting it to be a highly sensitive system among the available methods.
人类细胞中的主要信号通路与抗氧化防御系统有关。该系统的主要组成部分是转录因子，即红细胞 2 相关因子 2（NRF2）。它在氧化应激或抗氧化剂的刺激下，根据不同的细胞情况来调节该系统。因此，通过筛选策略检测 NRF2 的刺激，可能使我们能够发现 NRF2 相关信号通路的刺激剂。考虑到这一点，我们设计了一种包含 NRF2 反应元件的全细胞生物报告器，该元件插入到荧光素酶载体中，位于已去除启动子的荧光素酶基因的上游。该生物报告器可被 3H-1,2-二硫杂茂-3-硫酮（D3T）、丁基羟基茴香醚（BHA）和抗坏血酸等刺激物激活，这些刺激物作为抗氧化剂起作用。据观察，NRF2 蛋白识别的 NRF2 基因的调节区位于其编码区内部。该设计的生物报告器可以检测抗氧化剂的存在。它还在不同剂量的这些试剂中表现出显著的线性相关性，范围从 0.8 到 80 μM 的抗坏血酸、0.1 到 100 μM 的 D3T 和 0.1 到 100 μM 的 BHA。该检测系统被证明比实时 PCR 更灵敏，表明它在现有方法中是一种高度灵敏的系统。
