中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (31): 4990-4995.doi: 10.12307/2023.448

• 骨科植入物相关基础实验 Basic experiments of orthopedic implant • 上一篇    下一篇

Wnt蛋白生成抑制剂2干预破骨细胞的分化

廖紫芮,陈建权   

  1. 苏州大学医学部骨科研究所,江苏省苏州市  215000
  • 收稿日期:2022-04-11 接受日期:2022-07-14 出版日期:2023-11-08 发布日期:2023-01-31
  • 通讯作者: 陈建权,特聘教授,博士生导师,苏州大学医学部骨科研究所,江苏省苏州市 215000
  • 作者简介:廖紫芮,女,1997年生,浙江省龙泉市人,汉族,苏州大学医学部骨科研究所在读硕士,主要从事骨发育与再生研究。
  • 基金资助:
    国家自然科学基金(81974344),项目负责人:陈建权

Effect of inhibitor of Wnt production 2 on osteoclast differentiation

Liao Zirui, Chen Jianquan   

  1. Institute of Orthopedics, Suzhou Medical College, Soochow University, Suzhou 215000, Jiangsu Province, China
  • Received:2022-04-11 Accepted:2022-07-14 Online:2023-11-08 Published:2023-01-31
  • Contact: Chen Jianquan, Professor, Doctoral supervisor, Institute of Orthopedics, Suzhou Medical College, Soochow University, Suzhou 215000, Jiangsu Province, China
  • About author:Liao Zirui, Master candidate, Institute of Orthopedics, Suzhou Medical College, Soochow University, Suzhou 215000, Jiangsu Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81974344 (to CJQ)

摘要:


文题释义:

Wnt蛋白生成抑制剂2(IWP-2):是Wnt配体加工和分泌的抑制剂,靶向膜结合O-酰基转移酶porcupine(Porcn)并使其失活,从而选择性地抑制Porcn介导关键的Wnt配体棕榈酰化。
破骨细胞:是巨噬细胞系形成的巨大多核细胞,主要分布在骨质表面、骨内血管通道周围,行使骨吸收和释放矿物基质的功能,在骨发育和骨稳态中发挥重要作用。

背景:破骨细胞介导的骨吸收异常激活,导致骨量减少、骨微结构受损和恶化,是骨质疏松症的重要原因。Wnt/β-catenin信号通路可能是预防和治疗骨质疏松症的潜在靶点及工具,过度激活Wnt/β-catenin信号通路可以抑制破骨细胞分化。然而,Wnt/β-catenin信号通路在破骨细胞形成中的生理作用仍有待明确。
目的:探讨Wnt蛋白生成抑制剂2(the inhibitor of Wnt production 2,IWP-2)抑制Wnt/β-catenin信号通路对骨髓来源巨噬细胞增殖及分化能力的影响。
方法:①提取骨髓来源巨噬细胞,在0-40 μmol/L 的区间内设置浓度梯度,通过CCK-8实验评估不同浓度 IWP-2处理24,72,120 h后对骨髓来源巨噬细胞增殖的影响;②在体外建立经典的破骨细胞分化模型,用5,10 μmol/L IWP-2及等量溶剂处理经100 μg/L核因子κB受体激活因子配体诱导的骨髓来源巨噬细胞,收集0,1,3,5 d的蛋白,通过Western blot检测破骨分化过程中Wnt/β-catenin信号通路活力变化及 IWP-2对其的抑制作用;③用非毒性浓度(0,5,10 μmol/L)的 IWP-2处理经100 μg/L核因子κB受体激活因子配体诱导的骨髓来源巨噬细胞6 d,通过抗酒石酸酸性磷酸酶染色评估 IWP-2对破骨细胞分化的影响,实时荧光定量PCR检测破骨细胞分化相关特征基因NFATc1、Oscar、CTSK、Acp5、Mmp9、Dcstamp  mRNA的表达水平。

结果与结论:①不同浓度 IWP-2处理24 h,10 μmol/L IWP-2可促进骨髓来源巨噬细胞增殖;不同浓度 IWP-2处理72 h 和120 h,10 μmol/L及以下浓度的 IWP-2对骨髓来源巨噬细胞增殖无明显影响,而高浓度(20,30,40 μmol/L)IWP-2可抑制骨髓来源巨噬细胞增殖;②核因子κB受体激活因子配体刺激1,3,5 d,对照组Active β-catenin的蛋白表达水平逐渐升高,而5,10 μmol/L IWP-2处理组Active β-catenin的蛋白表达水平均受到抑制;③对照组可见许多大且细胞形态不规则的多核成熟破骨细胞形成,而 IWP-2处理组显示这些细胞的数量和大小呈剂量依赖性下降,表明 IWP-2可抑制破骨细胞分化;④破骨细胞分化相关特征基因 NFATc1、Oscar、CTSK、Acp5、Mmp9、Dcstamp mRNA表达水平经IWP-2处理后均下调;⑤提示IWP2可在体外抑制破骨细胞分化,其可能通过直接或间接抑制破骨细胞分化相关特征基因的表达,起到预防和治疗骨质疏松症的作用。

https://orcid.org/0000-0003-1579-538X (廖紫芮)

中国组织工程研究杂志出版内容重点:人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程

关键词: 骨质疏松症, Wnt/β-catenin信号通路, IWP-2, 骨髓来源巨噬细胞, 细胞增殖, 细胞分化

Abstract: BACKGROUND: Abnormal activation of osteoclast-mediated bone resorption leads to the loss of bone mass, damage and deterioration of bone microarchitecture, which is an important cause of osteoporosis. Wnt/β-catenin signaling pathway may be a potential target and tool for the prevention and treatment of osteoporosis. Overactivation of Wnt/β-catenin signaling pathway inhibits osteoclast differentiation. However, the physiological role of Wnt/β-catenin signaling pathway in osteoclast formation remains to be clarified.
OBJECTIVE: To investigate the effect of inhibitor of Wnt production 2 (IWP-2) on proliferation and differentiation of bone marrow-derived macrophages through inhibiting the Wnt/β-catenin signaling pathway.
METHODS: (1) Bone marrow-derived macrophages were extracted. Cell counting kit-8 assay was performed to assess the viability of bone marrow-derived macrophages after being treated with gradient concentrations from 0 to 40 μmol/L IWP-2 for 24, 72, or 120 hours. (2) Bone marrow-derived macrophages induced by 100 μg/L receptor activator of nuclear factor kappa B ligand (RANKL) were treated with 5 or 10 μmol/L IWP-2 and equivalent solvent, and precipitated proteins were collected at 0, 1, 3 and 5 days. The activity of Wnt/β-catenin signaling pathway and the inhibitory effect of IWP-2 on it during osteoclast differentiation were detected by western blot assay. (3) Bone marrow-derived macrophages induced by 100 μg/L RANKL were treated with non-toxic IWP-2 (0, 5, or 10 μmol/L) for 6 days. The effects of IWP-2 on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase staining. The mRNA expression of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were detected by real-time fluorescence quantitative PCR.
RESULTS AND CONCLUSION: (1) After treatment with different concentrations of IWP-2 for 24 hours, 10 μmol/L IWP-2 promoted the proliferation of bone marrow-derived macrophages. After treatment with different concentrations of IWP-2 for 72 and 120 hours, the viability of bone marrow-derived macrophages was not significantly affected by IWP-2 at a concentration of 10 μmol/L or less, while high concentrations of IWP-2 (20, 30, and 40 μmol/L) inhibited the proliferation of bone marrow-derived macrophages. (2) After 1, 3, and 5 days of RANKL stimulation, the protein expression of active β-catenin in the control group increased gradually, while that in the 5 and 10 μmol/L IWP-2 treatment group was significantly inhibited. (3) Many large and irregular TRAP-positive multinucleated mature osteoclasts were observed in the control group, while the number and size of the cells decreased in a dose-dependent manner in the IWP-2 treatment groups, suggesting that IWP-2 could inhibit osteoclast differentiation. (4) The expression levels of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were all down-regulated in a dose-dependent manner after IWP-2 treatment. (5) To conclude, IWP-2 can inhibit osteoclast differentiation in vitro and may play a role in the prevention and treatment of osteoporosis by directly or indirectly inhibiting the expression of osteoclast-elated genes.

Key words: osteoporosis, Wnt/β-catenin signaling pathway, IWP-2, bone marrow-derived macrophage, proliferation, differentiation

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