中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (17): 2643-2649.doi: 10.3969/j.issn.2095-4344.3518

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

淫羊藿苷通过提高自噬促进成骨细胞分化防治骨质疏松

姜  涛1，2，凌翠敏2，陈庆真3，杨冰璇1，林燕平1，邵  敏3   

  1. 1广州中医药大学，广东省广州市   510405；2广东省第二中医院，广东省广州市   510095；3广州中医药大学第三附属医院，广东省广州市  510240

  • 收稿日期:2020-03-19 修回日期:2020-03-24 接受日期:2020-05-19 出版日期:2021-06-18 发布日期:2021-01-08
  • 通讯作者: 邵敏，博士研究生导师，主任医师，广州中医药大学第三附属医院，广东省广州市 510240
  • 作者简介:姜涛，男，1989年生，汉族，湖南省长沙市人，广州中医药大学博士研究生，医师，主要从事骨质疏松与骨关节疾病研究。
  • 基金资助:
    广州市科技计划项目(201707010463)，项目负责人：邵敏；国家自然科学基金面上项目(81373654) ，项目负责人：邵敏；广东省自然科学基金项目(2015A030313351)，项目负责人：邵敏

Icariin prevents osteoporosis by activating autophagy and promoting osteoblast differentiation

Jiang Tao1, 2, Ling Cuimin2, Chen Qingzhen3, Yang Bingxuan1, Lin Yanping1, Shao Min3   

  1. 1Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou 510095, Guangdong Province, China; 3The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • Received:2020-03-19 Revised:2020-03-24 Accepted:2020-05-19 Online:2021-06-18 Published:2021-01-08
  • Contact: Shao Min, Doctoral supervisor, Chief physician, the Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • About author:Jiang Tao, MD candidate, Physician, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Guangdong Second Traditional Chinese Medicine Hospital, Guangzhou 510095, Guangdong Province, China
  • Supported by:
    Guangzhou Science and Technology Planning Project, No. 201707010463 (to SM); the National Natural Science Foundation of China, No. 81373654 (to SM); Guangdong Natural Science Foundation, No. 2015A030313351 (to SM)

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摘要：

文题释义：
淫羊藿苷：是中药淫羊藿的主要活性成分之一，具有补肾壮阳、抗衰老等功效，研究表明淫羊藿苷能够调节骨代谢，减缓骨丢失速度，防止骨质疏松的发生。
自噬：是一种通过清除受损细胞器、蛋白从而维持细胞存活的重要生理机制，研究表明自噬能够调控成骨细胞、破骨细胞、骨细胞的功能，对维持骨骼强度、预防骨质疏松的发生有重要的意义。

背景：淫羊藿苷是用于防治骨质疏松疗效比较确切的中药活性成分，但其具体的机制尚未完全明确。研究表明自噬能够参与调控骨代谢，淫羊藿苷是否能够通过改变自噬防治骨质疏松尚未可知。
目的：研究淫羊藿苷是否通过调节自噬促进成骨细胞分化防治骨质疏松。
方法：①体外实验：选取对数生长期小鼠前体成骨细胞株(MC3T3-E1)，用不同浓度的淫羊藿苷(10-6-10-3 mmol/L)干预，通过观察细胞活力、细胞凋亡率、碱性磷酸酶活性、矿化结节数量、成骨分化标志物表达水平确定淫羊藿苷的最佳作用浓度；用最佳浓度淫羊藿苷处理MC3T3-E1细胞，将细胞分为3组：对照组、淫羊藿苷组及淫羊藿苷+3-MA组(自噬抑制剂)，MDC染色检测自噬体数量，实时荧光定量聚合酶链反应检测成骨分化标志物及自噬相关基因的表达水平，免疫印迹法检测自噬相关蛋白的表达水平；②体内实验：通过构建去势大鼠模型，将动物分为4组：假手术组、病理模型组、淫羊藿苷组、戊酸雌二醇组，显微CT检测各组骨显微参数，免疫印迹法检测各组自噬相关蛋白表达水平。
结果与结论：①淫羊藿苷能促进成骨细胞的增殖以及分化，同时抑制细胞凋亡，以10-5 mmol/L 淫羊藿苷能力最强(P < 0.05)；②淫羊藿苷能够增加自噬体的数量，同时可上调成骨分化标志物及自噬相关基因、蛋白的表达(P < 0.05)，3-MA能够拮抗这些效应(P < 0.05)；③淫羊藿苷能够改善去势大鼠骨微结构相关参数，同时上调自噬相关蛋白的表达(P < 0.05)；④结果提示，淫羊藿苷能够通过提高自噬促进成骨细胞分化防治骨质疏松。
https://orcid.org/0000-0002-2837-6347(姜涛) 

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: 自噬, 基因, 淫羊藿苷, 成骨细胞, 分化, 骨, 大鼠, 实验

Abstract: BACKGROUND: Icariin is an effective active component of traditional Chinese medicine which is used to prevent osteoporosis, but the specific mechanism remains unclear. Existing studies have indicated that autophagy can participate in regulating bone metabolism; however, whether icariin can prevent osteoporosis by changing autophagy is unknown.  
OBJECTIVE: To investigate whether icariin can prevent osteoporosis by regulating autophagy and promoting osteoblast differentiation.
METHODS:  Mouse preosteoblast cell lines MC3T3-E1 in logarithmic phase were selected and treated by different concentrations of icariin (10-6 to 
10-3 mmol/L). Cell viability, apoptosis rate, alkaline phosphatase activity, the number of mineralized nodules and the expression level of osteogenic differentiation markers were observed in order to determine the optimal concentration of icariin. Then cells were treated by the optimal concentration of icariin and divided into three groups: control group, icariin group, icariin+3-methyladenine group. The number of autophagosomes, the expression level of osteogenic differentiation markers and autophagy related genes, the expression level of autophagy related proteins were evaluated by MDC staining, RT-qPCR and western blot assay, respectively. In an in vivo test, an ovariectomized rat model was established and all the rats were divided into four groups: sham group, ovariectomized group, icariin group, estradiol group. Micro-CT was used to detect the bone micro parameters, and western blot was used to detect the expression level of autophagy related proteins.
RESULTS AND CONCLUSION:  Icariin could promote the proliferation and differentiation of osteoblasts, and at the same time inhibit apoptosis of the cells. The maximum stimulatory effect of icariin was observed at the concentration of 10-5 mmol/L (P < 0.05). In addition, icariin could increase the number of autophagosomes and upregulate the expression of osteogenic differentiation markers and autophagy-related genes or proteins (P < 0.05), which were antagonized by 3-methyladenine (P < 0.05). Icariin could also improve the parameters of bone microstructure in ovariectomized rats and upregulate the expression of autophagy-related proteins (P < 0.05). All these findings indicate that icariin can prevent osteoporosis by activating autophagy and promoting osteoblast differentiation.

Key words: autophagy, gene, icariin, osteoblast, differentiation, bone, rat, experiment

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