Effect of Methysticin on osteoblast function under oxidative stress through Nrf2/HO-1 signaling pathway: in vitro study

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

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Artikel online veröffentlicht:
05. März 2021