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

Abstract

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.

Fig. 1. Nuclear factor E2-related factor 2 (Nrf2) cascade activation by 4-OI in osteoblasts.

Human osteoblastic OB-6 cells (a–g) or the primary murine osteoblasts (h, i) were treated with 4-OI (10/25 μM) or vehicle control (0.2% of DMSO, “Veh”) and cultured for applied time periods; Keap1–Nrf2 association was tested by co-immunoprecipitation (Co-IP assay) (a). Expression of listed proteins in cytosol fraction lysates and nuclei fraction lysates was tested by western blotting (b, d, g, h), with the expression of listed mRNAs tested by qPCR (c, f, i). The relative ARE reporter luciferase activity was tested as well (e). The morphology of murine osteoblasts with ALP staining is shown (h, the left panel). Expression of the listed proteins was quantified and normalized to the loading control (a, b, d, g, h). Quantified values were mean ± standard deviation (SD, n = 5). “n.s.” stands for no statistical difference (c). *P < 0.05 vs. “Veh” treatment. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (h).

Fig. 2. H₂O₂-induced oxidative injury was inhibited by 4-OI in osteoblasts.

Human osteoblastic OB-6 cells (a–c) or primary murine osteoblasts (d–f) were pretreated for 2 h with 4-OI (10/25 μM) or vehicle control (“Veh”), followed by H₂O₂ (400 μM) stimulation. Cells were further cultured for applied time periods, reactive oxygen species (ROS) production (tested by CellROX intensity, a, d), the GSH/GSSG ratio (b, e), and single-strand DNA (ssDNA) contents (ELISA OD, c, f) were tested. Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. cells with H₂O₂ stimulation but “Veh” pretreatment. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (a, d).

Fig. 3. H₂O₂-induced apoptosis is inhibited by 4-OI in osteoblasts.

Human osteoblastic OB-6 cells (a–e) or primary murine osteoblasts (f–i) were pretreated for 2 h with 4-OI (10/25 μM) or vehicle control (“Veh”), followed by H₂O₂ (400 μM) stimulation. Cells were further cultured for applied time periods; cell viability (CCK-8 OD, a, f), caspase-3 activity (b, g), and expression of listed proteins (western blotting assays, c) were tested; cell apoptosis was examined by nuclear TUNEL staining (d, h) and Annexin V FACS (e, i) assays, data were quantified. Expression of the listed proteins was quantified and normalized to the loading control (c). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. cells with H₂O₂ stimulation but “Veh” pretreatment. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (d).

Fig. 4. H₂O₂-induced programmed necrosis is inhibited by 4-OI in osteoblasts.

Human osteoblastic OB-6 cells (a–c) or the primary murine osteoblasts (d, e) were pretreated for 2 h with 4-OI (10/25 μM) or vehicle control (“Veh”), followed by H₂O₂ (400 μM) stimulation. Cells were further cultured for applied time periods; mitochondrial CyPD-ANT1-p53 association and their expression are shown (a); mitochondrial depolarization was tested by JC-1 green monomer fluorescence (b, d), and cell necrosis examined by quantifying medium LDH release (c, e). Expression of the listed proteins was quantified and normalized to the loading control (a). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. cells with H₂O₂ stimulation but “Veh” pretreatment. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (b).

Fig. 5. Forced overexpression of IRG1 activates nuclear factor E2-related factor (2Nrf2) signaling and protects osteoblasts from H₂O₂.

Stable OB-6 cells with the lentiviral IRG1 expression construct (“OE-IRG1” cells) or the empty vector (“Vec” cells) were established, expression of listed genes was tested by qPCR and western blotting analyses (a, c). The itaconate contents (b) and relative ARE reporter luciferase activity (d) are shown. “OE-IRG1” cells or “Vec” control OB-6 cell were treated with H₂O₂ (400 μM) and cultured for applied time periods; reactive oxygen species (ROS) contents (CellROX intensity, e), cell viability (CCK-8 OD, f), cell apoptosis (nuclear TUNEL staining assay, g) were tested; Mitochondrial depolarization was tested by JC-1 green monomers (h), and cell necrosis examined by quantifying medium LDH release (i). Expression of the listed proteins was quantified and normalized to the loading control (a, c). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. H₂O₂ stimulation in “Vec” cells. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (e, g, h).

Fig. 6. Nuclear factor E2-related factor 2 (Nrf2) cascade activation mediates 4-OI-induced osteoblast cytoprotection against H₂O₂.

Stable OB-6 cells with Nrf2 shRNA (“sh-Nrf2” cells) or the CRISPR/Cas9-Nrf2-KO-GFP construct (“ko-Nrf2” cells), as well as the control OB-6 cells with scramble control shRNA plus CRISPR/Cas9 empty vector (“sh-C+Cas9-C” cells), were established and cultured, expressions of listed genes are shown (a, b). Cells were pretreated for 2 h with 4-OI (25 μM), followed by H₂O₂ (400 μM) stimulation, and cultured for applied time periods. Cell viability (c) and apoptosis (d) were tested by CCK-8 and nuclear TUNEL staining assays, respectively. Stable OB-6 cells with the CRISPR/Cas9-Keap1-KO-GFP construct (“ko-Keap1” cells) and the control OB-6 cells with CRISPR/Cas9 empty vector (“Cas9-C” cells) were established and cultured. Cells were treated with or without 4-OI (25 μM) for another 6 h, expression of listed genes is shown (e, f). These cells were pretreated for 2 h with 4-OI (25 μM), followed by H₂O₂ (400 μM) stimulation, and cultured for applied time periods. Cell viability (g) and apoptosis (h) were tested similarly. Expression of the listed proteins was quantified and normalized to the loading control (a, e). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. ^#P < 0.05 vs. “sh-C+Cas9-C” cells or “Cas9-C” cells. “n.s.” stands for no significant difference (f–h). Experiments were repeated three times, with similar results obtained.