Introduction Bone healing is reported to be disturbed by excessive oxidative stress. Nuclear factor
erythroid 2-related factor 2 (Nrf2) is responsible for regulating antioxidant systems,
and it also has an important role in fracture healing. Our previous study showed that
Nrf2-knockout mice had reduced fracture callus compared with wild type mice, but the
effect of pharmacological Nrf2 induction on bone healing is still unknown. Given that
osteoblast function is generally essential for bone healing process, to investigate
the effect of Nrf2-inducer on osteoblasts under oxidative stress would lead to a future
therapeutic strategy for patients with refractory fracture healing. The purpose of
this in vitro study is to examine the effects of Nrf2-inducer methysticin on osteoblast
proliferation and mineralization under hydrogen peroxide (H2O2)-induced oxidative
stress condition.
Methods Murine MC3T3-E1 preosteoblasts were used for this study. Oxidative stress condition
was induced by various amount of H2O2 injection on cell culture medium. Methysticin,
widely used as a Nrf2-inducer, was used in this experiment. Heme oxygenase (HO-1)
expression, a Nrf2-target gene, was evaluated by PCR at the different term after methysticin
injection to detect the optimal timing of injection. Cell Proliferation was evaluated
by using total protein assay. Cell viability was evaluated by Apoptosis/Necrosis assay
kit. The mineralization of osteoblasts was obtained 21 days after the conversion from
normal medium to differentiation medium and was assessed by Alizarin-red staining.
The protein levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-6
during mineralization process were measured by ELISA.
Results A high amount of H2O2 (200μM) reduced 70 % of cell proliferation, while a low amount
of H2O2 (40μM) reduced only 16 % compared with non-H2O2 injection. Apoptosis/Necrosis
analysis showed that numerous apoptotic cells were observed after 200μM of H2O2 induction,
but the number of apoptotic cells was decreased after 200μM of H2O2 and methysticin
induction. HO-1 mRNA expression was significantly increased after methysticin injection
(a peak: 6 h after the injection). No mineralization was observed after 200μM of H2O2
induction, whereas mineralization was obtained after 40μM of H2O2 injection. With
methysticin injection, a slight mineralization was observed even under 200μM of H2O2
condition. Methysticin prevented cells from decreased release of VEGF and increased
IL6 level by H2O2 injection during mineralization process.
Discussion In our study, excessive H2O2 exposure caused osteoblast cell apoptosis and inhibited
mineralization through a decrease of VEGF but an increase IL-6 release, while methysticin
injection suppressed cell apoptosis and these markers modification through enhanced
HO-1 expression regardless of a slight improvement of mineralization on Alizarin-red
staining. These results indicate that pharmacological Nrf2 induction can have a potential
role to protect osteoblast function from oxidative stress through HO-1 signaling pathway.
Keywords Nrf2, bone healing, osteoblasts, HO-1, mineralization
Korrespondenzadresse Yusuke Kubo, Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, 52074
Aachen, Germany
E-Mail
ykubo@ukaachen.de
