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Influence of Nrf2 modulation on bone biomarkers in ovariectomized mice Introduction: The nuclear erythroid 2 p-45-related factor (Nrf2) controls gene expression of phase II detoxifying enzymes and oxidative-stress inducible proteins 1 . Oxidative stress regulates cellular functions including bone formation and resorption. In vitro studies have shown the effects of Nrf2 on osteoblast and osteoclast differentiation2. However, the role of this transcription factor in bone metabolism in vivo is not known. Objective: The aim of this study was to investigate the influence of Nrf2 modulation on bone biomarkers in ovariectomized mice. Methods: 25-30 week-old wild-type (Nrf2+/+) C57BL/6J female mice and Nrf2-deficient (Nrf2-/-) littermates were used in the present study. All mice were maintained in animal house with a 12-hour light/dark cycle, at 22ºC and free access to water and food. Ovariectomy (OVX) or sham-surgery was performed in each strain under deep anesthesia with isofluorane (1.5 minimum alveolar concentration) which was followed by subcutaneous injection of butorphanol (2 mg/kg). The study included 6 groups (n=14 per group): Nrf2+/+ OVX, Nrf2+/+ Sham, Nrf2-/- OVX, Nrf2-/- Sham and two groups of Nrf2+/+ mice that were administered with sulforaphane (50 mg/kg/day i.p.) from days 7 to 30: SFN-OVX and SFN-Sham. Blood was collected by retro-orbital puncture at day 30 after surgery, and animals were sacrificed by cervical dislocation. Hind paws were isolated for measurement of heme oxygenase-1 (HO-1) levels in their homogenates. Levels of bone formation markers alkaline phosphatase (ALP) and osteocalcin, and bone resorption markers type I collagen cross-linked C-telopeptide (CTX-I) and tartrate-resistant acid phosphatase isoform 5b (TRAP-5b), as well as receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) were measured in serum samples. Results were analysed by Student’s t-test. Results: Ovariectomy increased RANKL/OPG (0.26±0.03, p<0.01) and CTX-I (24.3±0.9 ng/ml, p<0.05) and reduced osteocalcin (17.2±0.6 ng/ml; p<0.05) and HO-1 (0.32±0.03, p<0.01) in Nrf2+/+ mice (Nrf2+/+ OVX group) compared to Nrf2+/+ Sham (0.09±0.01, 21.1±0.3 ng/ml, 20.8±0.4 ng/ml, 0.52±0.04, respectively). Nrf2+/+ Sham mice showed lower ALP (81.9±4.9 K.A. Units) levels than Nrf2-/- Sham mice (110.5±4.2 K.A. Units, p<0.01). Moreover, deficiency of Nrf2 increased levels of ALP (87.9±4.9 K.A. Units, p<0.05) and osteocalcin (20.5±1.1 ng/ml, p<0.05) and reduced RANKL/OPG (0.11±0.01, p<0.01) in ovariectomized mice compared to Nrf2+/+ OVX. On the other hand, induction of Nrf2 by sulforaphane in ovariectomized mice (SFN-OVX group) increased osteocalcin (21.5±0.6 ng/ml, p<0.01) and HO-1 (0.52±0.06, p<0.05) levels and decreased RANKL/OPG (0.15±0.02, p<0.05) and CTX-I (17.7±2.1 ng/ml, p<0.05) levels compared to Nrf2+/+ OVX. TRAP-5b levels were not modified by ovariectomy, Nrf2 deficiency or stimulation. Conclusion: Our study provides novel insights into the role of Nrf2 in bone metabolism in vivo. Nrf2 deficiency could positively regulate osteoblast differentiation; however, induction of Nrf2 by sulforaphane administration could inhibit osteoclast activity. Our findings also suggest that sulforaphane may be an effective agent to prevent bone loss. 1. Maicas, N.; Ferrandiz, M. L.; Brines, R.; Ibanez, L.; Cuadrado, A.; Koenders, M. I.; van den Berg, W. B.; Alcaraz, M. J. Antioxidants & Redox Signal. 2011, 15, 889-901.
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