Department of Molecular and Developmental Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan.
Japan Society for the Promotion of Science (JSPS), Tokyo 102-0083, Japan.
Int J Mol Sci. 2022 May 8;23(9):5243. doi: 10.3390/ijms23095243.
Antioxidant effects of soy-derived isoflavones are predicted to be mediated by the Keap1-Nrf2 pathway. Recently, we constructed an assay system to evaluate the antioxidant effects of dietary phytochemicals in zebrafish and revealed a relationship between these effects and the Keap1-Nrf2 pathway. In this study, we used this system to examine the antioxidant effects of seven isoflavones. Among those seven, equol showed strong antioxidant effects when arsenite was used as an oxidative stressor. The antioxidant effect of equol was also shown in Nrf2-mutant zebrafish , suggesting that this effect was not mediated by the Keap1-Nrf2 pathway. To elucidate this unidentified mechanism, the gene expression profiles of equol-treated larvae were analyzed using RNA-seq and qRT-PCR, while no noticeable changes were detected in the expression of genes related to antioxidant effects, except weak induction of Nrf2 target genes. Because is an amino acid-substitution mutant (Arg485Lue), we considered that the antioxidant effect of equol in this mutant might be due to residual Nrf2 activity. To examine this possibility, we generated an Nrf2-knockout zebrafish using CRISPR-Cas9 and analyzed the antioxidant effect of equol. As a result, equol showed strong antioxidant effects even in Nrf2-knockout larvae, suggesting that equol indeed upregulates antioxidant activity in zebrafish in an Nrf2-independent manner.
大豆异黄酮的抗氧化作用预计是通过 Keap1-Nrf2 通路介导的。最近，我们构建了一个评估膳食植物化学物质在斑马鱼中抗氧化作用的测定系统，并揭示了这些作用与 Keap1-Nrf2 通路之间的关系。在这项研究中，我们使用该系统来检测七种异黄酮的抗氧化作用。在这七种异黄酮中，当使用亚砷酸盐作为氧化应激物时，雌马酚表现出强烈的抗氧化作用。雌马酚的抗氧化作用也在 Nrf2 突变型斑马鱼中表现出来，表明该作用不是通过 Keap1-Nrf2 通路介导的。为了阐明这种未识别的机制，使用 RNA-seq 和 qRT-PCR 分析了雌马酚处理的幼虫的基因表达谱，除了 Nrf2 靶基因的弱诱导外，未检测到与抗氧化作用相关的基因表达的明显变化。因为 是一个氨基酸取代突变体（Arg485Lue），我们认为雌马酚在这个突变体中的抗氧化作用可能是由于残留的 Nrf2 活性。为了检验这种可能性，我们使用 CRISPR-Cas9 生成了 Nrf2 敲除斑马鱼，并分析了雌马酚的抗氧化作用。结果表明，即使在 Nrf2 敲除幼虫中，雌马酚也表现出强烈的抗氧化作用，表明雌马酚确实以 Nrf2 非依赖性方式在斑马鱼中上调抗氧化活性。
