中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (28): 4473-4479.doi: 10.12307/2023.531

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

汉防己甲素诱导自噬抑制破骨细胞的分化

张博文1，2，李  美1，张晋宁1， 杨天翔1，程萌旗3，陈德胜4   

  1. 宁夏医科大学，1临床医学院，2少数民族医药现代化教育部重点实验室，宁夏回族自治区银川市  750004；3上海交通大学附属第六人民医院关节外科，上海市  200233；4宁夏回族自治区人民医院骨科，宁夏回族自治区银川市  750004
  • 收稿日期:2022-08-10 接受日期:2022-09-21 出版日期:2023-10-08 发布日期:2023-01-29
  • 通讯作者: 陈德胜，博士，主任医师，教授，硕士生导师，宁夏回族自治区人民医院骨科，宁夏回族自治区银川市 750004
  • 作者简介:张博文，男，1995年生，湖北省武汉市人，汉族，宁夏医科大学在读硕士，主要从事骨科专业的研究。
  • 基金资助:
    国家自然科学基金项目(82060408)，项目负责人：陈德胜；宁夏回族自治区重点研发计划项目(2021BEG03049)，项目负责人：陈德胜

Tetrandrine inhibits osteoclast differentiation by inducing autophagy

Zhang Bowen1, 2, Li Mei1, Zhang Jinning1, Yang Tianxiang1, Cheng Mengqi3, Chen Desheng4   

  1. 1School of Clinical Medicine, 2Key Laboratory of Modernization of Minority Medicine, Ministry of Education, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Department of Joint Surgery, The Sixth Affiliated People‘s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China; 4Department of Orthopedics, People’s Hospital of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2022-08-10 Accepted:2022-09-21 Online:2023-10-08 Published:2023-01-29
  • Contact: Chen Desheng, MD, Chief physician, Master’s supervisor, Department of Orthopedics, People’s Hospital of Ningxia Hui Autonomous Region, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Zhang Bowen, Master candidate, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Key Laboratory of Modernization of Minority Medicine, Ministry of Education, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 82060408 (to CDS); Key R&D Project of Ningxia Hui Autonomous Region, No. 2021BEG03049 (to CDS)

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摘要：

文题释义：

自噬：是一种亚细胞生物行为，可由饥饿、氧化应激，理化伤害等多种因素激发。自噬过程利用溶酶体降解自身衰老、受损的细胞器和大分子等。总的来说，自噬是一种细胞对自身的保护机制并与临床中多种疾病的发生发展相联系。
汉防己甲素：即汉防己碱(Tetrandrine，Tet)，提取于防己科植物粉防己的块状根茎，主要药理作用有抗炎、逆转多重耐药性、诱导细胞凋亡等。也有不少关于汉防己甲素激发细胞自噬的相关研究，但更多的是将这一药理作用与肿瘤领域相结合。

背景：已有研究表明汉防己甲素可以通过激活AMPK信号通路诱导细胞自噬从而抑制肿瘤细胞增殖，但目前汉防己甲素诱导破骨细胞自噬和分化的相关研究甚少。
目的：探究汉防己甲素在不同浓度下对破骨细胞分化和自噬的影响，并分析诱导破骨细胞自噬能否起到抑制其分化的作用。
方法：①第一部分：使用重组小鼠巨噬细胞集落刺激因子和核因子κB受体活化因子配体两种细胞因子对小鼠RAW264.7巨噬细胞进行诱导，使其向破骨细胞转化。细胞诱导及药物干预分组如下：空白对照组(完全培养基)，阳性对照组(含两种细胞因子)，低、中、高剂量汉防己甲素干预组(阳性对照组分别添加0.1，0.5，1.0 μmol/L汉防己甲素)。抗酒石酸酸性磷酸酶染色法观察成熟破骨细胞数量变化；western blot法检测组织蛋白酶K(CTSK)、自噬微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和自噬底物泛素结合蛋白(p62)的蛋白相对表达量；RT-PCR检测CTSK、LC3-Ⅱ和p62的mRNA相对表达量；丹酰尸胺(MDC)染色观察诱导过程中破骨细胞中自噬小体的数量变化。②第二部分：选取抑制破骨细分化效果最明显的汉防己甲素浓度(1.0 μmol/L)进行回复性验证。各组处理如下：阳性对照组(含两种细胞因子)，自噬抑制剂组(巴佛洛霉素处理)，药物干预组(汉防己甲素干预)，药物干预+自噬抑制剂组。抗酒石酸酸性磷酸酶染色法观察成熟破骨细胞数量变化；Western blot法检测CTSK的蛋白相对表达量。

结果与结论：①一定浓度汉防己甲素能够对破骨细胞的分化起抑制作用，并且抑制作用随药物浓度升高而提升；②汉防己甲素在一定浓度下能够激发破骨细胞分化过程中自噬标志物的表达及减少自噬特异性底物累积，并在破骨细胞分化过程中可刺激自噬小体形成；③使用自噬抑制剂后，汉防己甲素抑制破骨细胞分化的作用受到显著影响；④综上，一定浓度的汉防己甲素能够通过激活自噬抑制破骨细胞的分化。

https://orcid.org/0000-0002-9691-0601(张博文) 

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: 汉防己甲素, 破骨细胞, 自噬, 骨溶解, 微管相关蛋白1轻链3-Ⅱ, CTSK

Abstract: BACKGROUND: Previous studies have shown that tetrandrine can inhibit the proliferation of tumor cells by activating AMPK signaling pathway to induce autophagy. However, there are few studies on tetrandrine induced autophagy and differentiation of osteoclasts.  
OBJECTIVE: To explore the effect of tetrandrine at different concentrations on autophagy during osteoclast differentiation, and analyze whether inducing autophagy of osteoclasts can inhibit their differentiation.  
METHODS: Part one: Recombinant mouse macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand cytokines were used to induce mouse RAW264.7 macrophages into osteoclasts. Mouse RAW264.7 macrophages were divided into five groups: blank group (complete medium), positive control group (treated by two cytokines), and low-, medium-, and high-concentration drug groups (treated by two cytokines and added 0.1, 0.5, and 1.0 μmol/L tetrandrine, respectively). Tartrate resistant acid phosphatase staining method was used to detect the number of mature osteoclasts. Western blot assay and RT-PCR were utilized to detect the relative expression levels of proteins of Cathepsin K, autophagy microtubule-associated protein 1 light chain 3-II, and autophagy substrate ubiquitin-binding protein (p62) at protein and mRNA levels, respectively. Monodansyl cadaverine staining was used to detect the number of autophagosomes in osteoclasts. Part two: The concentration of tetrandrine (1.0 μmol/L) with the best inhibitory effect on osteoclast differentiation was selected for regression verification. Cells were divided into positive control group (treated by two cytokines), autophagy inhibitor group (bafloamycin), drug intervention group (tetrandrine), and drug intervention+authophage inhibitor group. Tartrate resistant acid phosphatase staining was used to detect the number of mature osteoclasts. Western blot assay was utilized to detect the relative protein expression level of Cathepsin K. 
RESULTS AND CONCLUSION: Tetrandrine at a certain concentration inhibited the differentiation of osteoclasts and the inhibitory effect increased with the increase of drug concentration. Tetrandrine at a certain concentration could stimulate the expression of autophagy markers, reduce the accumulation of autophagy specific substrates during osteoclast differentiation, and stimulate the formation of autophagosomes during osteoclast differentiation. After using autophagy inhibitor, the effect of tetrandrine on inhibiting osteoclast differentiation was significantly affected. In conclusion, tetrandrine at a certain concentration can inhibit the differentiation of osteoclasts by activating autophagy.

Key words: tetrandrine, osteoclast, autophagy, osteolysis, microtubule-associated protein 1 light chain 3-II, CTSK

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