Calcified Tissue International

, Volume 105, Issue 4, pp 446–457 | Cite as

Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis via NADPH Oxidase-Interleukin 1β and 6 Pathway in Osteocyte-like Cells

  • Masakazu Notsu
  • Ippei KanazawaEmail author
  • Ayumu Takeno
  • Ken-ichiro Tanaka
  • Toshitsugu Sugimoto
Original Research


Homocysteine (Hcy) increases oxidation and inflammation; however, the mechanism of Hcy-induced bone fragility remains unclear. Because selective estrogen modulators (SERMs) have an anti-oxidative effect, SERMs may rescue the Hcy-induced bone fragility. We aimed to examine whether oxidative stress and pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 are involved in the Hcy-induced apoptosis of osteocytes and whether bazedoxifene (BZA) inhibits the detrimental effects of Hcy. We used mouse osteocyte-like cell lines MLO-Y4-A2 and Ocy454. Apoptosis was examined by DNA fragmentation ELISA and TUNEL staining, and gene expression was evaluated by real-time PCR. Hcy 5 mM significantly increased expressions of NADPH oxidase (Nox)1, Nox2, IL-1β, and IL-6 as well as apoptosis in MLO-Y4-A2 cells. Nox inhibitors, diphenyleneiodonium chloride and apocynin, significantly suppressed Hcy-induced IL-1β and IL-6 expressions. In contrast, an IL-1β receptor antagonist and an IL-6 receptor monoclonal antibody had no effects on Hcy-induced Nox1 and Nox2 expressions, but significantly rescued Hcy-induced apoptosis. BZA (1 nM–1 μM) and 17β estradiol 100 nM significantly rescued Hcy-induced apoptosis, while an estrogen receptor blocker ICI 182,780 reversed the effects of BZA and 17β estradiol. BZA also rescued Hcy-induced apoptosis of Ocy454 cell, and ICI canceled the effect of BZD. Moreover, BZA significantly ameliorated Hcy-induced expressions of Nox1, Nox2, IL-1β, and IL-6, and ICI canceled the effects of BZA on their expressions. Hcy increases apoptosis through stimulating Nox 1 and Nox 2-IL-1β and IL-6 expressions in osteocyte-like cells. BZA inhibits the detrimental effects of Hcy on osteocytes via estrogen receptor.


Homocysteine Bazedoxifene Osteocyte NADPH oxidase Interleukin-1β Interleukin-6 





Bazedoxifene acetate


NADPH oxidase


Diabetes mellitus


Type 2 diabetes mellitus


Bone mineral density


α-minimum essential medium


Selective estrogen receptor modulators


Diphenyleneiodonium chloride





This study had funding support from Pfizer. Pfizer did not affect the study protocol, interpretation of the results, or discussion. Authors’ roles: MN and IK were responsible for designing and conducting the study. MN performed the experiments and analyzed the data. AT and KT contributed equipment/materials. MN and IK wrote the manuscript. TS reviewed and edited the manuscript. All authors approved the final version. IK takes responsibility for the integrity of the data analysis. The authors thank Keiko Nagira for technical assistance.


IK and TS have received lecture fees from Pfizer.

Compliance with Ethical Standards

Conflict of interest

Masakazu Notsu, Ippei Kanazawa, Ayumu Takeno, Ken-ichiro Tanaka, and Toshitsugu Sugimoto declare that they have no competing interests.

Human and Animal Rights

This article does not contain any studies with human participants or animals performed by any of the authors.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Internal Medicine 1Shimane University Faculty of MedicineIzumoJapan

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