Four-octyl itaconate (4-OI) is the cell-permeable derivative of itaconate that can activate Nrf2 signaling by alkylating Keap1’s cysteine residues. Here, we tested the potential effect of 4-OI on hydrogen peroxide (H₂O₂)-induced oxidative injury in osteoblasts. In OB-6 cells and primary murine osteoblasts, 4-OI was able to activate Nrf2 signaling cascade and cause Keap1–Nrf2 disassociation, Nrf2 protein stabilization, cytosol accumulation, and nuclear translocation. 4-OI also augmented antioxidant-response element reporter activity and promoted expression of Nrf2-dependent genes (HO1, NQO1, and GCLC). Pretreatment with 4-OI inhibited H₂O₂-induced reactive oxygen species production, cell death, and apoptosis in osteoblasts. Furthermore, 4-OI inhibited H₂O₂-induced programmed necrosis by suppressing mitochondrial depolarization, mitochondrial cyclophilin D-ANT1 (adenine nucleotide translocase 1)-p53 association, and cytosol lactate dehydrogenase release in osteoblasts. Ectopic overexpression of immunoresponsive gene 1 (IRG1) increased endogenous itaconate production and activated Nrf2 signaling cascade, thereby inhibiting H₂O₂-induced oxidative injury and cell death. In OB-6 cells, Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout blocked 4-OI-induced osteoblast cytoprotection against H₂O₂. Conversely, forced Nrf2 activation, by CRISPR/Cas9-induced Keap1 knockout, mimicked 4-OI-induced actions in OB-6 cells. Importantly, 4-OI was ineffective against H₂O₂ in Keap1-knockout cells. Collectively, 4-OI efficiently activates Nrf2 signaling to inhibit H₂O₂-induced oxidative injury and death of osteoblasts.

四辛基衣康酸酯 (4-OI) 是衣康酸酯的细胞渗透性衍生物，可以通过将 Keap1 的半胱氨酸残基烷基化来激活 Nrf2 信号传导。在这里，我们测试了 4-OI 对过氧化氢 (H ₂ O ₂ ) 诱导的成骨细胞氧化损伤的潜在影响。在 OB-6 细胞和原代小鼠成骨细胞中，4-OI 能够激活 Nrf2 信号级联并导致 Keap1-Nrf2 解离、Nrf2 蛋白稳定、细胞质积累和核易位。4-OI 还增强了抗氧化反应元件报告基因的活性并促进了 Nrf2 依赖基因（HO1、NQO1和GCLC）的表达。4-OI 预处理抑制了 H ₂ O ₂诱导成骨细胞中活性氧的产生、细胞死亡和细胞凋亡。此外，4-OI通过抑制线粒体去极化、线粒体亲环蛋白 D-ANT1（腺嘌呤核苷酸转位酶 1）-p53 结合和成骨细胞中细胞质乳酸脱氢酶的释放来抑制 H ₂ O ₂诱导的程序性坏死。免疫反应基因 1 (IRG1) 的异位过表达增加了内源性衣康酸的产生并激活了 Nrf2 信号级联反应，从而抑制了 H ₂ O ₂诱导的氧化损伤和细胞死亡。在 OB-6 细胞中，Nrf2 沉默或 CRISPR/Cas9 诱导的 Nrf2 敲除阻断了 4-OI 诱导的成骨细胞对 H ₂ O _{2 的}保护作用. 相反，通过 CRISPR/Cas9 诱导的 Keap1 敲除强制 Nrf2 激活，模拟了 OB-6 细胞中 4-OI 诱导的作用。重要的是，4-OI 对Keap1 基因敲除细胞中的 H ₂ O ₂无效。总的来说，4-OI 有效地激活 Nrf2 信号传导以抑制 H ₂ O ₂诱导的成骨细胞氧化损伤和死亡。