Abstract
An ultra-large structure-based virtual screening has discovered iKeap1 as a direct Keap1 inhibitor that can efficiently activate Nrf2 signaling. We here tested its potential effect against hydrogen peroxide (H2O2)-induced oxidative injury in osteoblasts. In primary murine and human osteoblasts, iKeap1 robustly activated Nrf2 signaling at micromole concentrations. iKeap1 disrupted Keap1-Nrf2 association, causing Nrf2 protein stabilization, cytosol accumulation and nuclear translocation in murine and human osteoblasts. The anti-oxidant response elements (ARE) activity and transcription of Nrf2-ARE-dependent genes (including HO1, NQO1 and GCLC) were increased as well. Significantly, iKeap1 pretreatment largely ameliorated H2O2-induced reactive oxygen species production, lipid peroxidation and DNA damage as well as cell apoptosis and programmed necrosis in osteoblasts. Moreover, dexamethasone- and nicotine-induced oxidative injury and apoptosis were alleviated by iKeap1. Importantly, Nrf2 shRNA or CRISPR/Cas9-induced Nrf2 knockout completely abolished iKeap1-induced osteoblast cytoprotection against H2O2. Conversely, CRISPR/Cas9-induced Keap1 knockout induced Nrf2 cascade activation and mimicked iKeap1-induced cytoprotective actions in murine osteoblasts. iKeap1 was ineffective against H2O2 in the Keap1-knockout murine osteoblasts. Collectively, iKeap1 activated Nrf2 signaling cascade to inhibit H2O2-induced oxidative injury and death of osteoblasts.
Highlights
In the pathogenesis of osteoporosis or osteonecrosis, reactive oxygen species (ROS) overproduction will cause oxidative injury to osteoblasts, leading to sustained bone injury [1,2,3]
We found that this novel Keap1 inhibitor activated Nuclear factor E2-related factor 2 (Nrf2) signaling to inhibit H2O2-induced oxidative injury and death of osteoblasts
RESULTS iKeap1 activates Nrf2 cascade in murine and human osteoblasts First, we examined whether iKeap1 could activate Nrf2 signaling in osteoblasts
Summary
In the pathogenesis of osteoporosis or osteonecrosis, reactive oxygen species (ROS) overproduction will cause oxidative injury to osteoblasts, leading to sustained bone injury [1,2,3]. Hydrogen peroxide (H2O2) was added to cultured osteoblasts/ osteoblastic cells to mimic oxidative injury. It is a cellular model of osteoblast oxidative injury [4,5,6,7,8,9,10]. H2O2 stimulation in osteoblasts/osteoblastic cells should induce profound oxidative injury, lipid peroxidation, protein denaturation, and significant DNA damage. These events will together cause death of osteoblasts [5,6,7,8, 10]. The unstimulated Nrf protein stays in cytosol and directly binds to Keap (Kelch-like ECH-associated protein 1)
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