Department of Cardiology, Huai'an First People's Hospital, Nanjing Medical University, China.
Department of Critical Care Medicine, Changshu No.2 People's Hospital, Changshu, China.
Biochem Biophys Res Commun. 2021 Oct 15;574:110-117. doi: 10.1016/j.bbrc.2021.08.024. Epub 2021 Aug 9.
Nuclear factor E2-related factor 2 (Nrf2) activation could efficiently protect myocardial cells from oxygen glucose deprivation/re-oxygenation (OGDR). An ultra-large structure-based virtual screening has discovered iKeap1 as a novel, direct and potent Keap1 inhibitor. Here we found that iKeap1 efficiently activated Nrf2 signaling in H9c2 myocardial cells and primary murine myocardiocytes. iKeap1 induced Keap1-Nrf2 disassociation, cytosol Nrf2 protein stabilization and nuclear translocation. The antioxidant response element (ARE) activity and expression of Nrf2 cascade genes (HO1, NQO1 and GCLC) were increased in iKeap1-treated myocardial cells. In H9c2 cells and murine myocardiocytes, iKeap1 potently inhibited OGDR-induced oxidative injury by inhibiting reactive oxygen species (ROS) production, mitochondrial depolarization, lipid peroxidation and DNA damage. In addition, OGDR-induced myocardial cell death and apoptosis were largely ameliorated after pretreatment with the novel Keap1 inhibitor. Significantly, in H9c2 cells iKeap1-induced myocardial cytoprotection against OGDR was abolished with Nrf2 silencing or knockout (using CRISPR/Cas9 method). Moreover, CRISPR/Cas9-induced Keap1 knockout led to constitutive activation of Nrf2 cascade and inhibited OGDR-induced oxidative injury. Importantly, iKeap1 was unable to further protect Keap1-knockout H9c2 cells from OGDR. Together, iKeap1 activated Nrf2 signaling to protect myocardial cells from OGDR-induced oxidative injury and cell death.
核因子 E2 相关因子 2（Nrf2）的激活可以有效地保护心肌细胞免受氧葡萄糖剥夺/再氧合（OGDR）的损伤。一项基于超大结构的虚拟筛选发现，iKeap1 是一种新型的、直接的、有效的 Keap1 抑制剂。在这里，我们发现 iKeap1 可以有效地激活 H9c2 心肌细胞和原代鼠心肌细胞中的 Nrf2 信号通路。iKeap1 诱导 Keap1-Nrf2 解离、细胞质 Nrf2 蛋白稳定和核转位。抗氧化反应元件（ARE）活性和 Nrf2 级联基因（HO1、NQO1 和 GCLC）的表达在 iKeap1 处理的心肌细胞中增加。在 H9c2 细胞和鼠心肌细胞中，iKeap1 通过抑制活性氧（ROS）产生、线粒体去极化、脂质过氧化和 DNA 损伤，有效地抑制 OGDR 诱导的氧化损伤。此外，新型 Keap1 抑制剂预处理后，OGDR 诱导的心肌细胞死亡和凋亡得到了很大程度的改善。值得注意的是，在 H9c2 细胞中，iKeap1 诱导的 OGDR 心肌细胞保护作用被 Nrf2 沉默或敲除（使用 CRISPR/Cas9 方法）所消除。此外，CRISPR/Cas9 诱导的 Keap1 敲除导致 Nrf2 级联的组成性激活，并抑制 OGDR 诱导的氧化损伤。重要的是，iKeap1 不能进一步保护 OGDR 的 Keap1 敲除 H9c2 细胞。总之，iKeap1 通过激活 Nrf2 信号通路来保护心肌细胞免受 OGDR 诱导的氧化损伤和细胞死亡。
