中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (8): 1173-1179.doi: 10.12307/2022.220

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

左归丸干预卵巢摘除骨质疏松模型小鼠骨形态发生蛋白2信号通路的变化

李  微1，朱汉民2，王  鑫1，高  雪1，崔  婧1，刘雨昕1，黄树明3   

  1. 湖北文理学院，1基础医学院，2人事处，湖北省襄阳市   441053；3黑龙江中医药大学中医药研究院，黑龙江省哈尔滨市   150040
  • 收稿日期:2021-03-26 修回日期:2021-04-01 接受日期:2021-05-16 出版日期:2022-03-18 发布日期:2021-11-02
  • 通讯作者: 黄树明，博士，教授，博士生导师，黑龙江中医药大学中医药研究院，黑龙江省哈尔滨市 150040
  • 作者简介:李微，女，1988年生，黑龙江省绥棱县人，汉族，2018年黑龙江中医药大学毕业，博士，讲师，主要从事中医基础理论-肾主骨方面的研究。
  • 基金资助:
    湖北省教育厅科研计划项目(B2020137)：项目负责人：李微，课题名称：左归丸通过雌激素受体介导 BMP-2/Runx2/Osrtrix信号通路调控骨质疏松症的分子机制

Effect of Zuogui Wan on bone morphogenetic protein 2 signaling pathway in ovariectomized osteoporosis mice

Li Wei1, Zhu Hanmin2, Wang Xin1, Gao Xue1, Cui Jing1, Liu Yuxin1, Huang Shuming3   

  1. 1Basic Medical College, 2Personnel Office, Hubei University of Arts and Science, Xiangyang 441053, Hubei Province, China; 3Institute of Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang Province, China
  • Received:2021-03-26 Revised:2021-04-01 Accepted:2021-05-16 Online:2022-03-18 Published:2021-11-02
  • Contact: Huang Shuming, MD, Professor, Doctoral supervisor, Institute of Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang Province, China
  • About author:Li Wei, MD, Lecturer, Basic Medical College, Hubei University of Arts and Science, Xiangyang 441053, Hubei Province, China
  • Supported by:
    the Scientific Research Project of Hubei Provincial Department of Education, No. B2020137 (to LW)

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摘要：

文题释义：
骨质疏松：是由于多种原因导致的骨密度和骨质量下降，骨微结构破坏，造成骨脆性增加，从而容易发生骨折的全身性骨病。此文涉及的骨质疏松是由于卵巢摘除后，小鼠体内雌激素急速撤退导致骨质疏松发生。
卵巢摘除：卵巢摘除后会使体内雌激素急速撤退，雌激素缺乏是导致骨质疏松的主要诱因，可以通过这种手术方式模拟女性绝经期期间体内激素变化，建立绝经后骨质疏松小鼠模型。
背景：绝经后骨质疏松症在中老年女性中属于高发疾病，严重影响其生活质量，发现并探索出一种有效的防治手段是非常必要的。中医可通过“肾主骨”理论选用补肾药治疗绝经后骨质疏松症，但其分子机制尚不清楚。
目的：观察左归丸对骨形态发生蛋白2/Runx-2/Osterix信号通路的影响。
方法：采用卵巢摘除的方法建立绝经后骨质疏松症小鼠模型，然后将造模成功的84只小鼠分为模型组、假手术组、雌二醇组、左归丸高、中、低(0.936，0.468，0.234 g/L)剂量组、阻断剂组，每组12只。左归丸高、中、低剂量组给予相应质量浓度的左归丸水煎液；假手术组、模型组给予等体积的生理盐水；雌二醇组给予雌二醇水溶液；阻断剂组给予ICI182780+左归丸高剂量水煎液，持续灌胃8周。采用免疫组化和Western Blot法检测左归丸干预后卵巢摘除骨质疏松症模型小鼠胫骨和股骨中骨形态发生蛋白信号通路的关键蛋白骨形态发生蛋白2、Runx-2和Osterix的表达；通过ICI182780阻断雌激素受体后，检测骨形态发生蛋白通路中蛋白表达的变化。
结果与结论：①免疫组化结果显示，与模型组小鼠相比，左归丸高、中剂量均可以明显提高模型组小鼠骨形态发生蛋白2的表达水平    (P < 0.01，P < 0.05)；左归丸高、中、低剂量组Runx-2的表达水平相比差异均有显著性意义(P < 0.01)；②免疫印迹实验结果显示，与模型组小鼠相比，左归丸高、中剂量均可以明显提高模型组小鼠骨形态发生蛋白2的表达水平(P < 0.05)；左归丸高、中、低剂量组Runx-2的表达水平均显著高于模型组(P < 0.01，P < 0.05，P < 0.05)；左归丸高、中剂量组Osterix的表达水平均显著高于模型组(P < 0.01，P < 0.05)；③ICI182780干预后，与左归丸高剂量组相比，阻断剂组小鼠胫骨骨形态发生蛋白2和Runx-2蛋白的表达水平显著降低(P < 0.05，P < 0.01)；阻断剂组小鼠股骨中骨形态发生蛋白2、Runx-2和Osterix的表达水平均显著降低(P < 0.01)；④提示左归丸可以通过雌激素受体激活骨形态发生蛋白2/Runx-2/Osterix信号通路进而参与骨代谢过程，实现骨保护的作用。

https://orcid.org/0000-0001-7714-1184 (李微) 

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: 左归丸, 骨质疏松症, 卵巢摘除, 骨形态发生蛋白2, Runx-2, Osterix

Abstract: BACKGROUND: Postmenopausal osteoporosis is a high-incidence disease in middle-aged and elderly women, which seriously affects the quality of life. It is very necessary to discover and develop an effective prevention and treatment method. Traditional Chinese medicine can treat postmenopausal osteoporosis with tonifying kidney medicines based on the theory of “kidney governs bone,” but the molecular mechanism is still unclear.
OBJECTIVE: To observe the effect of Zuogui Wan on bone morphogenic protein 2 (BMP-2)/Runx-2/Osterix signaling pathway. 
METHODS: The postmenopausal osteoporosis model was established in mice by ovarian removal. Then the successfully modeled mice were divided into model group, sham operation group, estradiol group, high-, medium- and low-dose Zuogui Wan (0.936, 0.468, 0.234 g/L) groups, and blocker group, with 12 mice per group. In the high-, medium- and low-dose Zuogui Wan groups, the corresponding concentrations of Zuogui Wan decoction were given. Sham operation group and model group were given the equal volume of normal saline. Estradiol group was given estradiol aqueous solution. The blocker group was given ICI182780 plus high-dose Zuogui Wan decoction. Intervention in each group was given via gavage for 8 continuous weeks. Immunohistochemistry and western blot were used to detect the expression of key proteins BMP-2, Runx-2 and Osterix in the tibia and femur of ovariectomized osteoporosis mice after intervention by Zuogui Wan. Changes of protein expressions in the BMP pathway were detected after blocking estrogen receptor using ICI182780.
RESULTS AND CONCLUSION: Immunohistochemical findings indicated that compared with the model group, the high and medium doses of Zuogui Wan could significantly increase the expression of BMP-2 in ovariectomized osteoporosis mice (P < 0.01, P < 0.05); the expression of Runx-2 in the medium- and low-dose Zuogui Wan groups was significantly different (P < 0.01). Western blot results showed that compared with the model group, the high and middle doses of Zuogui Wan significantly increased the expression of BMP-2 in ovariectomized osteoporosis mice (P < 0.05); the expression of Runx-2 was significantly higher in the high-, medium- and low-dose Zuogui Wan groups (P < 0.01, P < 0.05, P < 0.05); and the expression of Osterix was significantly higher in the high- and medium-dose Zuogui Wan groups (P < 0.01, P < 0.05). After ICI182780 intervention, compared with the high-dose group, the expression of BMP-2 and Runx-2 in the mouse tibia was significantly reduced in the blocker group (P < 0.05, P < 0.01) and the expression of BMP-2, Runx-2 and Osterix in the mouse femoral bones was significantly reduced in the blocker group (P < 0.01). To conclude, Zuogui Wan can participate in the regulation of bone metabolism by increasing the expression of important proteins in the BMP/Runx-2/Osterix signaling pathway in ovariectomized osteoporosis mice, thereby achieving the protection of bones.

Key words: Zuogui Wan, osteoporosis, ovariectomy, bone morphogenic protein 2, Runx-2, Osterix

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