Department of Neurosurgery, The First People's Hospital of Kunshan, Jiangsu University, Suzhou, China.
Department of Endocrinology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
Cell Commun Signal. 2018 Nov 15;16(1):81. doi: 10.1186/s12964-018-0294-2.
Four-octyl itaconate (OI), the itaconate's cell-permeable derivative, can activate Nrf2 signaling via alkylation of Keap1 at its cysteine residues. The current study tested the potential neuroprotective function of OI in hydrogen peroxide (HO)-treated neuronal cells.
SH-SY5Y neuronal cells and epigenetically de-repressed (by TSA treatment) primary murine neurons were treated with OI and/or HO. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Neuronal cell death was tested by the LDH and trypan blue staining assays. Apoptosis was tested by TUNEL and Annexin V assays.
In SH-SY5Y neuronal cells and primary murine neurons, OI activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, as well as expression of Nrf2-regulated genes (HO1, NQO1 and GCLC) and ninjurin2 (Ninj2). Functional studies showed that OI attenuated HO-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage as well as neuronal cell death and apoptosis. shRNA-mediated knockdown, or CRISPR/Cas9-induced knockout of Nrf2 almost abolished OI-induced neuroprotection against HO. Keap1 is the primary target of OI. Keap1 knockout by CRISPR/Cas9 method mimicked and abolished OI-induced actions in SH-SY5Y cells. Introduction of a Cys151S mutant Keap1 in SH-SY5Y cells reversed OI-induced Nrf2 activation and anti-HO neuroprotection.
OI activates Keap1-Nrf2 signaling to protect SH-SY5Y cells and epigenetically de-repressed primary neurons from HO in vitro.
辛二烯基琥珀酸（OI）是一种可穿透细胞的琥珀酸衍生物，可以通过半胱氨酸残基对 Keap1 的烷基化来激活 Nrf2 信号通路。本研究检测了 OI 在过氧化氢（HO）处理神经元细胞中的潜在神经保护作用。
用 OI 和/或 HO 处理 SH-SY5Y 神经元细胞和经表观遗传去抑制（TSA 处理）的原代小鼠神经元。通过 Western blot 检测和实时定量 PCR 分析检测 Nrf2 通路基因。通过 LDH 和台盼蓝染色试验检测神经元细胞死亡。通过 TUNEL 和 Annexin V 试验检测细胞凋亡。
在 SH-SY5Y 神经元细胞和原代小鼠神经元中，OI 激活了 Nrf2 信号通路，导致 Keap1-Nrf2 解离、Nrf2 蛋白稳定和核转位，以及 Nrf2 调节基因（HO1、NQO1 和 GCLC）和 ninjurin2（Ninj2）的表达。功能研究表明，OI 可减轻 HO 诱导的活性氧（ROS）产生、脂质过氧化和 DNA 损伤以及神经元细胞死亡和凋亡。Nrf2 的 shRNA 介导的敲低或 CRISPR/Cas9 诱导的敲除几乎消除了 OI 对 HO 的神经保护作用。Keap1 是 OI 的主要靶点。CRISPR/Cas9 方法敲除 Keap1 可模拟并消除 OI 在 SH-SY5Y 细胞中诱导的作用。在 SH-SY5Y 细胞中引入 Cys151S 突变型 Keap1 可逆转 OI 诱导的 Nrf2 激活和抗 HO 神经保护作用。
OI 通过激活 Keap1-Nrf2 信号通路，在体外保护 SH-SY5Y 细胞和表观遗传去抑制的原代神经元免受 HO 的损害。
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