| 芒柄花素抑制RANKL诱导破骨细胞分化的实验研究 |
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Received:April 08, 2019
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| 作者 | Author | 单位 | Unit | E-Mail |
| 洪一波 |
HONG Yi-bo |
南京中医药大学附属苏州市中医医院, 江苏 苏州 215009 |
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| 姜宏 |
JIANG Hong |
南京中医药大学附属苏州市中医医院, 江苏 苏州 215009 |
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| 王建伟 |
WANG Jian-wei |
南中医药大学附属无锡市中医医院, 江苏 无锡 214071 |
Wuxi Hospital Affiliated to Nanjing University of Traditional Chinese Medicine, Wuxi 214071, Jiangsu, China |
diaphysis@qq.com |
| 俞鹏飞 |
YU Peng-fei |
南京中医药大学附属苏州市中医医院, 江苏 苏州 215009 |
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| 尤武林 |
YOU Wu-lin |
南中医药大学附属无锡市中医医院, 江苏 无锡 214071 |
Wuxi Hospital Affiliated to Nanjing University of Traditional Chinese Medicine, Wuxi 214071, Jiangsu, China |
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| 期刊信息：《中国骨伤》2020年33卷，第1期，第64-70页 |
| DOI：10.3969/j.issn.1003-0034.2020.01.012 |
| 基金项目:江苏省青年医学重点人才项目（编号：QNRC2016253）；无锡市青年医学重点人才项目（编号：QNRC011）；苏州市中医医院博士青年课题（编号：YQN2018003） |
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目的：建立RANKL诱导的破骨细胞体外研究模型，阐述活血化瘀中药鸡血藤有效组分芒柄花素（formononetin，FO）调控小鼠骨髓单核-巨噬细胞（BMMs）向破骨细胞分化和功能的影响，探讨其抑制破骨细胞分化的分子机制。
方法：取20只4～6周龄清洁级C57/BL6小鼠，雌雄各10只，体重（20±2） g，无菌条件下分离出股骨和胫骨内BMMs，用α-MEM培养基进行体外培养和增殖。BMMs在加入M-CSF和不同浓度的芒柄花素（5～50 μM）分别培养4 d后进行细胞增殖与毒性的CCK8检测。将生长状态良好的BMMs依次加入M-CSF和RANKL诱导破骨细胞分化，对照组无特殊处理，DMSO对照组加入DMSO溶剂，各观察组分别加入不同浓度芒柄花素（1～20 μM），分别进行培养6 d后进行抗酒石酸酸性磷酸酶（TRAP）染色，对破骨细胞的进行计数和统计分析。分别在破骨细胞培养的1、2 d收取总蛋白和磷酸化蛋白，Western blot检测破骨细胞分化中关键转录因子NFATc1和c-Fos的表达以及磷酸化蛋白ERK表达；在培养的4 d提取RNA，Real-Time PCR检测破骨细胞相关基因CTSK、TRAP、MMP9和Car2的活性。
结果：CCK8检测结果提示芒柄花素能够剂量依赖性地抑制BMMs的活性，在≤20 μM的安全浓度范围内对BMMs细胞生长无明显毒性效应（P=0.278>0.05）。TRAP染色结果发现芒柄花素在（1～20 μM）浓度范围内能够剂量依赖性的抑制破骨细胞的生成，尤其是10 μM能够显著抑制破骨细胞的生成（P=0.000<0.05）。Western blot检测表明芒柄花素（10 μM）能显著抑制破骨细胞分化关键蛋白NFATc1和c-Fos的表达，而对磷酸化蛋白ERK的表达未见明显的作用。在破骨细胞功能上，Real-Time PCR检测芒柄花素（10 μM）能著抑制破骨细胞功能相关基因CTSK（P=0.000<0.05）、TRAP（P=0.000<0.05）、MMP9（P=0.000<0.05）和Car2（P=0.000<0.05）的表达。
结论：鸡血藤有效组分芒柄花素能够抑制原代骨髓单核-巨噬细胞向破骨细胞增殖和分化，并下调破骨细胞骨吸收功能相关蛋白和基因的表达，可能是其防治股骨头坏死中骨破坏及塌陷的机制之一。 |
| [关键词]:芒柄花素 骨髓-单核巨噬细胞 破骨细胞生成 抗酒石酸酸性磷酸酶 |
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| Experimental study on the inhibition of Formononetin on the differentiation of osteoclasts induced by RANKL |
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Abstract:
Objective: To establish the in vitro study model of osteoclasts induced by RANKL,to elaborate the effect of formononetin(FO),an effective component of Caulis Spatholobi,on the differentiation and function of bone marrow mononuclear macrophages(BMMs) into osteoclasts,and to explore the molecular mechanism of its inhibition.
Methods: The BMMs in femur and tibia were isolated from 20 clean C57/BL6 mice of 4 to 6 weeks old,10 males and 10 females,each weighing (20±2) g. The BMMs in femur and tibia were cultured and proliferated in vitro with α-MEM medium. BMMs were cultured with M-CSF and different concentrations of anthocyanin (5 to 50 μm) respectively for 4 days,and CCK8 of cell proliferation and toxicity was detected. BMMs in good growth condition were added to M-CSF and RANKL to induce osteoclast differentiation in turn. There was no special treatment in the control group. DMSO was added to the control group with DMSO solvent. Each observation group was added with different concentrations of awnasin (1 to 20 μm). After 6 days of culture,the osteoclasts were counted and statistically analyzed. The expression of NFATc1,c-Fos and ERK,the key transcription factors in osteoclast differentiation,were detected by Western blot,RNA was extracted at 4 days,and the activity of ctsk,trap,MMP9 and Car2 were detected by real time PCR.
Results: CCK8 test results showed that awnstein could inhibit the activity of BMMs in a dose-dependent manner,and had no significant toxic effect on the growth of bmms within the safe concentration range of ≤ 20 μM(P=0.278>0.05). The results of trap staining showed that awnstein could inhibit osteoclast production in a dose-dependent manner in the concentration range of (1 to 20 μM),especially in 10 μM(P=0.000<0.05). Western blot showed that 10 μ m could significantly inhibit the expression of NFATc1 and c-Fos,but not the expression of ERK. In terms of osteoclast function,the expression of ctsk(P=0.000<0.05),trap(P=0.000<0.05),MMP9(P=0.000<0.05) and Car2(P=0.000<0.05) related to osteoclast function were detected by real time PCR.
Conclusion: The effective component of Caulis Spatholobi can inhibit the proliferation and differentiation of primary mononuclear macrophages into osteoclasts,and down regulate the expression of osteoclast bone resorption related proteins and genes,which may be one of the mechanisms of its prevention and treatment of bone destruction and collapse in osteonecrosis of femoral head. |
| KEYWORDS:Formononetin Bone marrow macrophages Osteoclastogenesis Tartrate resistant acid phosphatase |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 洪一波,姜宏,王建伟,俞鹏飞,尤武林.芒柄花素抑制RANKL诱导破骨细胞分化的实验研究[J].中国骨伤,2020,33(1):64~70 |
| 英文格式: | HONG Yi-bo,JIANG Hong,WANG Jian-wei,YU Peng-fei,YOU Wu-lin.Experimental study on the inhibition of Formononetin on the differentiation of osteoclasts induced by RANKL[J].zhongguo gu shang / China J Orthop Trauma ,2020,33(1):64~70 |
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