中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (17): 2636-2641.doi: 10.12307/2022.527

• 骨组织构建 bone tissue construction • 上一篇    下一篇

射干苷抑制破骨细胞分化的作用与机制

黄发义1,刘予豪2,周  驰2,黄浩然1,陈伟坚3,何  伟2,王海彬2   

  1. 1广州中医药大学第一临床医学院,广东省广州市   510405;2广州中医药大学第一附属医院,广东省广州市   510405;3广州中医药大学附属广州市正骨医院,广东省广州市   510405
  • 收稿日期:2020-12-24 修回日期:2020-12-29 接受日期:2021-05-30 出版日期:2022-06-18 发布日期:2021-12-24
  • 通讯作者: 王海彬,博士后,教授,广州中医药大学第一附属医院,广东省广州市 510405
  • 作者简介:黄发义,男,1995年生,广东省徐闻县人,汉族,广州中医药大学在读硕士,主要从事股骨头坏死、骨质疏松、骨性关节炎研究。
  • 基金资助:
    2021年广东省自然科学基金面上项目(2021A1515011484),项目负责人:刘予豪;2021年广州市科技计划项目·基础与应用基础研究项目 (202102020930) ,项目负责人:刘予豪;国家自然科学基金面上项目(81774339,82074462),项目负责人:王海彬

Belamcandin inhibits osteoclast differentiation: its role and mechanism

Huang Fayi1, Liu Yuhao2, Zhou Chi2, Huang Haoran1, Chen Weijian3, He Wei2, Wang Haibin2   

  1. 1First Clinical School of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 3Guangzhou Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Received:2020-12-24 Revised:2020-12-29 Accepted:2021-05-30 Online:2022-06-18 Published:2021-12-24
  • Contact: Wang Haibin, MD, Professor, First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • About author:Huang Fayi, Master candidate, First Clinical School of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Supported by:
    Guangdong Provincial Natural Science Foundation (General Project), No. 2021A1515011484 (to LYH); Guangzhou Science and Technology Project · Basic and Applied Basic Research Project, No. 202102020930 (to LYH); the National Natural Science Foundation of China, Nos. 81774339 and 82074462 (both to WHB)

摘要:

文题释义:
射干苷(Tectoridin):也叫鸢尾苷,是一种植物雌激素,可激活雌激素和甲状腺激素受体。射干苷通过雌激素受体依赖性基因组途径和 GPR30 依赖性非基因组途径发挥雌激素作用,其具有结构多样性、毒性较低及来源广泛等特点。
活化T细胞核因子c1(nuclear factor of activated T-cells cytoplasmic 1,NFATc1):是破骨细胞分化过程中最关键的转录调节分子,其可以直接调节破骨细胞相关的特异性基因的转录表达;同时接受MAPK、核因子κB、钙离子、活性氧等多种信号通路的调节,触发其激活和自身进行自我扩增,从而促进破骨细胞的分化。
破骨细胞分化:破骨细胞是一种直径达100 mm的多核巨噬细胞,成熟的破骨细胞由骨髓单核细胞分化而来,其分化成熟过程可以分为几个阶段:单核细胞增殖期,前体细胞融合、增殖期,破骨细胞形成期,破骨细胞骨吸收功能期;骨髓细胞在巨噬细胞集落刺激因子的诱导下形成骨髓单核细胞,随后在巨噬细胞集落刺激因子和核因子κB受体活化因子的诱导下,骨髓单核细胞逐渐分化,形成成熟破骨细胞,成熟破骨细胞抗酒石酸酸性磷酸酶染色呈阳性。

背景:核因子κB受体激活因子配体介导的Calcium/NFATc1通路激活在破骨细胞分化中发挥重要作用。射干苷可通过激活雌激素受体达到雌激素样作用,但目前尚缺乏其对破骨细胞分化及功能影响的相关报道。
目的:探讨射干苷对破骨细胞的作用及其机制。
方法:体外培养小鼠骨髓巨噬细胞分化成熟后种入96孔板中(每孔为6×103个),分别加入5,10,20,30,40 μmol/L的射干苷,观察对破骨细胞分化抑制作用,筛选出作用最强的射干苷浓度。分别以不同浓度的射干苷干预核因子κB受体激活因子配体诱导的破骨细胞体外分化过程,进行抗酒石酸酸性磷酸酶染色,观察射干苷对破骨细胞形成和功能的影响。通过钙离子振荡实验、Western Blot及RT-qPCR等实验,观察Calcium/NFATc1信号通路中上下游基因和蛋白的表达情况。实验方案经广州中医药大学第一临床医学院动物实验伦理委员会批准。
结果与结论:①射干苷可抑制破骨细胞形成,且抑制作用呈剂量依赖性,40 μmol/L的射干苷对破骨细胞分化抑制作用最强;②射干苷可抑制与破骨细胞形成相关的Acp5(TRAcP)基因以及c-Fos、Integrin β3蛋白的表达,同时抑制与骨吸收功能相关的Ctsk、基质金属蛋白酶9基因以及转录蛋白的表达;③射干苷可抑制钙离子信号通道的开放频率及强度、抑制Ctr(Calcitonin Receptor)、Nfatc1基因及转录蛋白的表达;④结果说明:射干苷可通过核因子κB受体激活因子配体诱导的钙离子信号通道抑制Nfatc1基因及转录蛋白的表达,从而抑制破骨细胞的形成及骨吸收功能。
缩略语:核因子κB受体激活因子配体:receptor activator of nuclear factor kappa B ligand,RANKL;核因子κB受体活化因子:receptor activator of nuclear factor kappa B,RANK;活化T细胞核因子 c1:nuclear factor of activated T-cells cytoplasmic 1,NFATc1;抗酒石酸酸性磷酸酶:tartrate resistant acid phosphatase,TRACP

https://orcid.org/0000-0002-0977-485X (黄发义) 

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: 射干苷, 破骨细胞, 钙离子, NFATc1, 细胞分化

Abstract: BACKGROUND: Receptor activator of nuclear factor kappa B ligand can mediate the activation of calcium/nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) pathway, which is crucial for osteoclast differentiation. Belamcandin can exert estrogen-like effects by activating estrogen receptors; however, less is reported on its effects on the differentiation and function of osteoclasts.
OBJECTIVE: To explore the effect of Belamcandin on osteoclasts and its mechanism.
METHODS: Mature mouse bone marrow macrophages cultured in vitro were differentiated and seeded into 96-well plates (6×103 cells per well), followed by inhibitory interventions with 5, 10, 20, 30, 40 μmol/L Belamcandin. Osteocyte differentiation was then observed to screen out the optimal concentration of Belamcandin with the strongest effect. Different concentrations of Belamcandin were used to deal with the differentiation of osteoclasts induced by nuclear factor κB receptor activator ligands. Tartrate-resistant acid phosphatase staining was used to observe the effects of Belamcandin on the formation and function of osteoclasts. Expression of upstream and downstream genes and proteins in the Calcium/NFATc1 signaling pathway was observed through calcium ion oscillation experiments, western blot assay and RT-qPCR detection. An approval was obtained from the Animal Experiment Ethics Committee of the First Clinical Medical College of Guangzhou University of Chinese Medicine.
RESULTS AND CONCLUSION: Belamcandin inhibited the formation of osteoclasts in a dose-dependent manner, and 40 μmol/L Belamcandin had the strongest potential to inhibit osteoclast differentiation. Belamcandin could inhibit the mRNA expression of Acp5 (tartrate resistant acid phosphatase) gene related to the inhibition of osteoclast formation and the protein expression of c-Fos and Integrin β3, while inhibiting the expression of Ctsk, matrix metalloproteinase 9 gene, and transcription protein related to the inhibition of bone resorption function. Belamcandin could inhibit the opening frequency and intensity of calcium ion signal channels, and restrain the expression of Calcitonin receptor, Nfatc1 gene and transcription protein. To conclude, Belamcandin can inhibit the expression of Nfatc1 gene and transcription protein through the calcium ion signaling pathway induced by nuclear factor κB receptor activator ligand, thereby inhibiting osteoclast formation and bone resorption.

Key words: Belamcandin, osteoclasts, calcium ions, NFATc1, cell differentiation

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