Four-octyl itaconate activates Nrf2 cascade to protect osteoblasts from hydrogen peroxide-induced oxidative injury

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 (H 2 O 2 )-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 H 2 O 2 -induced reactive oxygen species production, cell death, and apoptosis in osteoblasts. Furthermore, 4-OI inhibited H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 . 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 H 2 O 2 in Keap1-knockout cells. Collectively, 4-OI efficiently activates Nrf2 signaling to inhibit H 2 O 2 -induced oxidative injury and death of osteoblasts.