Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress

Abstract

MIND4-17 is a recently developed NF-E2-related factor 2 (Nrf2) activator, which uniquely causes Nrf2 disassociation from Keap1. Here, we showed that pretreatment with MIND4-17 significantly inhibited hydrogen peroxide (H₂O₂)-induced viability reduction of primary osteoblasts and OB-6 osteoblastic cells. Meanwhile, MIND4-17 inhibited both apoptotic and non-apoptotic osteoblast cell death by H₂O₂. MIND4-17 treatment induced Keap1-Nrf2 disassociation, causing Nrf2 stabilization, accumulation and nuclear translocation in osteoblasts, leading to transcription of several Nrf2-dependent genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC). Additionally, MIND4-17 largely attenuated H₂O₂-reactive oxygen species (ROS) production, lipid peroxidation and DNA damages. Nrf2 knockdown by targeted short hairpin RNA (shRNA) exacerbated H₂O₂-induced cytotoxicity in OB-6 osteoblastic cells, and nullified MIND4-17-mediated cytoprotection against H₂O₂. Meanwhile, Keap1 shRNA took over MIND4-17's actions and protected OB-6 cells from H₂O₂. Together, MIND4-17 activates Nrf2 signaling and protects osteoblasts from H₂O₂.

Keywords: Keap; MIND4-17; Nrf2; osteoblasts; oxidative stress.

Figure 1. MIND4-17 protects human osteoblasts from hydrogen peroxide

OB-6 human osteoblastic cells (A-D), the primary human osteoblasts (E) or the primary murine osteoblasts (F) were pretreated for 1 hour with applied concentration of MIND4-17, following by the hydrogen peroxide (“H₂O₂”) treatment, cells were further cultured for additional 48 hours in the conditional medium; Cell viability (CCK-8 assay, A, C, E and F) and cell death (LDH release assay, B and D) were tested. Data were presented as mean (n=5) ± standard deviation (SD). “C” stands for medium treatment control (Same for all figures). ^*p<0.05 vs. “C” cells. ^#p<0.05 vs. H₂O₂ only group. Experiments in this figure were repeated for four times, and similar results were obtained.

Figure 2. MIND4-17 inhibits H₂O₂-induced apoptotic and non-apoptotic cell death of osteoblasts

OB-6 human osteoblastic cells (A-E), the primary human osteoblasts (F) or the primary murine osteoblasts (G) were pretreated for 1 hour with MIND4-17 (3 μM), following by hydrogen peroxide (“H₂O₂”, 200μM) treatment, cells were then cultured for the indicated time period and were subjected to assays mentioned in the text to examine apoptotic and non-apoptotic cell death. Data were presented as mean (n=5) ± standard deviation (SD). ^*p<0.05 vs. “C” cells. ^#p<0.05 vs. H₂O₂ only group. Experiments in this figure were repeated for three times, and similar results were obtained.

Figure 3. MIND4-17 activates Nrf2 signaling in osteoblasts

OB-6 human osteoblastic cells were treated with MIND4-17 (3 μM) for applied time; The association between Nrf2 and Keap1 was examined by the co-immunoprecipitation (“Co-IP”) assay (A, left panel); Expressions of the indicated proteins in total cell lysates (A, right panel, and D) as well as in the nuclear lysates (B, Lamin-B was a marker protein) were presented; mRNA expressions of listed genes were tested by quantitative real-time PCR (“qRT-PCR”) assay (C). Data were presented as mean (n=5) ± standard deviation (SD). ^*p<0.05 vs. “C” cells. Experiments in this figure were repeated for three times, and similar results were obtained.

Figure 4. MIND4-17 alleviates H₂O₂-induced oxidative stress in osteoblasts

OB-6 human osteoblastic cells (A-C), the primary human osteoblasts (D) or the primary murine osteoblasts (E) were pretreated for 1 hour with MIND4-17 (3 μM), following by hydrogen peroxide (“H₂O₂”, 200μM) treatment, cells were then cultured for the indicated time; Relative ROS production (DCFH-DA fluorescence intensity, A, D and E), lipid peroxidation (TBAR intensity, B)and DNA damages (p-γ-H2AX ratio, C). Data were presented as mean (n=5) ± standard deviation (SD). ^*p<0.05 vs. “C” cells. ^#p<0.05 vs. H₂O₂ only group. Experiments in this figure were repeated for three times, and similar results were obtained.

Figure 5. Nrf2 activation is required for MIND4-17-mediated cytoprotection

OB-6 cells, stably expressing lentiviral scramble non-sense control shRNA (“sh-scr”) or Nrf2 shRNA (“sh-Nrf2, sequence -1/−2”) (A-E), as well as lentiviral Keap1 shRNA (“sh-Keap1”) (F-J), were treated with MIND4-17 (3 μM) for 3 hours, expressions of listed proteins were presented (A and F); mRNA expressions of listed genes were also shown (B, C, G and H); Cells were further treated with/out hydrogen peroxide (“H₂O₂”, 200 μM) for indicated time period, cell viability (CCK-8 assay, D and I) and apoptosis (Histone DNA ELISA assay, E and J) were also tested. For H₂O₂ experiments, MIND4-17 (3 μM) were pretreated before H₂O₂ for 1 hour. For all the assays, the exactsame number of viable cells with the applied shRNA was initially plated into each well. Data were presented as mean (n=5) ± standard deviation (SD). ^#p<0.05. Experiments in this figure were repeated for three times, and similar results were obtained.