二甲双胍对斑马鱼骨骼发育及损伤修复的机制研究

doi:10.16288/j.yczz.21-250

遗传 ›› 2022, Vol. 44 ›› Issue (1): 68-79.doi: 10.16288/j.yczz.21-250

二甲双胍对斑马鱼骨骼发育及损伤修复的机制研究

贾婷婷¹(), 雷蕾¹, 吴歆媛¹, 蔡顺有², 陈艺璇¹, 薛钰¹()

1. 闽南师范大学,福建省菌类活性物质工程技术研究中心,漳州 363000
2. 闽南师范大学,化学化工与环境学院,福建省现代分离分析重点实验室,漳州 363000

收稿日期:2021-07-13 修回日期:2021-09-13 出版日期:2022-01-20 发布日期:2021-12-03
通讯作者: 薛钰 E-mail:948450471@qq.com;xueyu0614@163.com
作者简介:贾婷婷,在读硕士研究生,专业方向：化学生物学。E-mail: 948450471@qq.com
基金资助:
福建省自然科学基金面上项目编号(2020J01823);福建省自然科学基金面上项目编号(2019J01744);闽南师范大学培育项目编号(MSPY202101);福建省教育厅中青年教师教育科研项目资助编号(JAT190357)

Study on the mechanism of metformin on zebrafish skeletal development and damage repair

Tingting Jia¹(), Lei Lei¹, Xinyuan Wu¹, Shunyou Cai², Yixuan Chen¹, Yu Xue¹()

1. The Engineering Technological Center of Fungus Active Substances of Fujian Province, Minnan Normal University, Zhangzhou 363000, China
2. Key Laboratory of Modern Analytical Science and Separation Technology of Fujian Province, School of Chemistry, Chemical Engineering, and Environment, Minnan Normal University, Zhangzhou 363000, China

Received:2021-07-13 Revised:2021-09-13 Online:2022-01-20 Published:2021-12-03
Contact: Xue Yu E-mail:948450471@qq.com;xueyu0614@163.com
Supported by:
Supported by the Natural Science Foundation of Fujian Province Nos(2020J01823);Supported by the Natural Science Foundation of Fujian Province Nos(2019J01744);Minnan Normal University Cultivation Project No(MSPY202101);Educational Research Project for Young and Middle-aged Teachers of Fujian Provincial Department of Education No(JAT190357)

摘要/Abstract

摘要：

二甲双胍(metformin, MET)是治疗糖尿病的一线药物,对骨骼疾病也有一定的治疗效果,但具体作用机制尚不明确。本研究利用斑马鱼(Danio rerio)构建骨质疏松模型,通过荧光观察、骨骼染色、半定量PCR、原位杂交及ELISA等技术方法,探究MET对斑马鱼骨骼发育及损伤修复的作用机制。首先通过胚胎致死率、骨骼矿化及钙化程度确定了MET的工作浓度是0.1%,该浓度MET对斑马鱼胚胎和幼鱼的骨骼发育均有显著的促进作用,可通过增强骨骼调控基因的mRNA水平和蛋白质水平实现。进一步利用枸橼酸铁铵(ferric ammonium citrate, FAC)和硝基还原酶/甲硝唑(nitroreductase/metronidazole, NTR/MTZ)系统构建斑马鱼体外和体内骨质疏松模型,并利用MET进行恢复实验,结果表明MET能显著修复FAC或MTZ诱导引起的骨骼矿化面积减小、脊椎钙化减少、成骨分化减弱等骨质疏松表型,通过促进成骨细胞再生、增强成骨细胞标记物(sp7、ALP)表达和抑制破骨细胞标记物(ctsk、mmp9、TRAP)的活性发挥修复作用。最后,通过检测Bmp信号成员的表达水平变化,初步证明MET不仅可以增强Bmp的mRNA和蛋白表达,还可通过激活Bmp下游信号通路促进斑马鱼骨骼发育和损伤修复。综合本研究的实验结果表明,MET作为治疗糖尿病专用药的同时,对斑马鱼骨骼发育也有促进作用,且对骨质疏松症具有显著的修复功效,为老药新用提供了新的研究方向和实验支撑。

关键词: 二甲双胍, 斑马鱼, 骨质疏松, 损伤修复, Bmp

Abstract:

Metformin (MET) is a well-known first-line drug used to treat diabetes. However, its therapeutic effect and the underlying mechanism in treatment of skeletal diseases are still unclear. In this study, we used the zebrafish osteoporosis model to explore the function and mechanism of metformin in zebrafish bone development and damage repair through fluorescence observation, bone staining, semi-quantitative PCR, in situ hybridization and ELISA assays. Firstly, the working concentration of MET was determined to be 0.1% through embryonic lethality, bone mineralization and calcification. At this concentration, MET enhanced the bone development of zebrafish embryos and juveniles through up-regulation of bone regulatory factors at the mRNA and protein levels. Furthermore, we used ferric ammonium citrate (FAC) and NTR/MTZ system to build zebrafish osteoporosis models in vitro and in vivo, followed by MET treatment, and the experimental results showed that MET can restore the osteoporotic phenotypes, such as the decreased bone mineralization area, reduced spine calcification, and weakened osteogenic differentiation induced by FAC or MTZ, and it functioned through promoting osteoblast regeneration, enhancing the expression of osteoblast markers (sp7, ALP), and inhibiting the activities of osteoclast markers (ctsk, mmp9, TRAP). Finally, we detected the expression levels of Bmp signaling components, and our preliminary data showed that MET can not only enhance the transcriptional and protein expression levels of Bmp, but also promote zebrafish bone development and damage repair by activating Bmp downstream signaling. Taken together, our results show that in addition to treating diabetes, MET can also promote zebrafish bone development and has a significant repair effect on osteoporosis, which will provide new research directions and experimental support for the new applications of old drugs.

Key words: metformin (MET), zebrafish, osteoporosis, damage rapair, Bmp

贾婷婷, 雷蕾, 吴歆媛, 蔡顺有, 陈艺璇, 薛钰. 二甲双胍对斑马鱼骨骼发育及损伤修复的机制研究[J]. 遗传, 2022, 44(1): 68-79.

Tingting Jia, Lei Lei, Xinyuan Wu, Shunyou Cai, Yixuan Chen, Yu Xue. Study on the mechanism of metformin on zebrafish skeletal development and damage repair[J]. Hereditas(Beijing), 2022, 44(1): 68-79.

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二甲双胍对斑马鱼骨骼发育及损伤修复的机制研究

Study on the mechanism of metformin on zebrafish skeletal development and damage repair

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