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Molecules 2017, 22(12), 2068; doi:10.3390/molecules22122068

Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization

1
Department of Biological Science & Technology College of Medicine, I-Shou University, Kaohsiung 82445, Taiwan
2
Department of Chemistry, Chinese Culture University, Taipei 11114, Taiwan
3
Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan
4
Center for Biomedical Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan
5
Center for Nanotechnology, Chung Yuan Christian University, Chung-Li 32023, Taiwan
*
Author to whom correspondence should be addressed.
Received: 13 September 2017 / Revised: 10 November 2017 / Accepted: 22 November 2017 / Published: 27 November 2017
(This article belongs to the Section Medicinal Chemistry)
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Abstract

Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. The mammalian system is considered too time-consuming, expensive and complex to perform high-throughput drug screening. There is a gap between in vitro cell-based models and the in vivo mammalian models. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for disease-based drug screening. In this study, zebrafish embryos were incubated with small molecular compounds that potentially affected bone mineralization in microplates. Two compounds of alendronate and dorsomorphin were used as positive and negative controls, respectively. The level of osteogenic mineralization was measured and quantified by using ImageJ software with fluorescent calcein-staining images. Among twenty-four tested compounds from the kinase inhibitor library, we identified two compounds, pentamidine and BML-267, which showed increased embryonic mineralization; while six compounds, RWJ-60475, levamisole HCL, tetramisole HCL, fenvalerate, NSC-663284, and BML-267ester, were inhibitory to bone mineralization. In addition, real time quantitative PCR (RT-qPCR) was performed to evaluate the biological pathways involved in bone metabolism at the molecular level. We confirmed that alendronate enhanced the level of bone mineralization by inhibiting osteoclast-related genes. In summary, our research established a simple method to screen potential bone metabolic drugs and to perform mechanism analysis for bone mineralization in vivo. View Full-Text
Keywords: drug screening; bone mineralization; osteoclast; calcein; zebrafish drug screening; bone mineralization; osteoclast; calcein; zebrafish
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Chen, J.-R.; Lai, Y.-H.; Tsai, J.-J.; Hsiao, C.-D. Live Fluorescent Staining Platform for Drug-Screening and Mechanism-Analysis in Zebrafish for Bone Mineralization. Molecules 2017, 22, 2068.

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