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

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Abstract

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.

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Abbreviations

Hcy:

Homocysteine

BZA:

Bazedoxifene acetate

Nox:

NADPH oxidase

DM:

Diabetes mellitus

T2DM:

Type 2 diabetes mellitus

BMD:

Bone mineral density

α-MEM:

α-minimum essential medium

SERM:

Selective estrogen receptor modulators

DPI:

Diphenyleneiodonium chloride

Apo:

Apocinin

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Acknowledgements

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.

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IK and TS have received lecture fees from Pfizer.

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Authors and Affiliations

  1. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan

    Masakazu Notsu, Ippei Kanazawa, Ayumu Takeno, Ken-ichiro Tanaka & Toshitsugu Sugimoto

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  1. Masakazu Notsu
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  2. Ippei Kanazawa
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Correspondence to Ippei Kanazawa.

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

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Notsu, M., Kanazawa, I., Takeno, A. et al. Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis via NADPH Oxidase-Interleukin 1β and 6 Pathway in Osteocyte-like Cells. Calcif Tissue Int 105, 446–457 (2019). https://doi.org/10.1007/s00223-019-00580-7

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