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J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (6): 571-577    DOI: 10.3785/j.issn.1008-9292.2017.12.01
    
Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway
WEI Zhenlong1(),SHI Wengui2,CHEN Keming2,ZHOU Jian2,WANG Minggang1,*()
1. College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
2. Institute of Orthopedic Research, Lanzhou General Hospital of PLA, Lanzhou 730050, China
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Abstract  

Objective: To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. Methods: The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. Results: Real time RT-PCR showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related genes in icaritin group were significantly increased (P<0.05 or P<0.01); After AMD3100 treatment, the relative expression of CXCR4 gene was decreased (P<0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all P<0.01), but were decreased after AMD3100 was added (all P<0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all P<0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all P<0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. Conclusion: Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.



Key wordsOsteoblasts/drug effects      Icariin/pharmacology      Chemokines, CXC/metabolism      Receptors, CXCR4/metabolism      Cell differentiation      Signal transduction      Mice      Cells, cultured     
Received: 07 July 2017      Published: 25 December 2017
CLC:  R681  
  R966  
Corresponding Authors: WANG Minggang     E-mail: weizhenlong1990@163.com;mgwang@163.com
Cite this article:

WEI Zhenlong,SHI Wengui,CHEN Keming,ZHOU Jian,WANG Minggang. Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway. J Zhejiang Univ (Med Sci), 2017, 46(6): 571-577.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2017.12.01     OR     http://www.zjujournals.com/med/Y2017/V46/I6/571


淫羊藿素通过CXCR4/SDF-1信号通路促进小鼠成骨细胞成熟和矿化

目的: 研究淫羊藿素是否通过CXC趋化因子受体4(CXCR4)/基质细胞衍生因子1(SDF-1)信号通路促进小鼠成骨细胞MC3T3-E1成熟和矿化。方法: 将体外培养的MC3T3-E1细胞分为空白对照组、AMD3100组、淫羊藿素组、淫羊藿素加AMD3100组。药物处理24 h时,采用实时定量RT-PCR和蛋白质印迹法检测CXCR4、SDF-1和成骨相关基因和蛋白的表达;药物处理3、6 d时,采用碱性磷酸酶(ALP)试剂盒检测ALP的活性;药物处理14 d时,采用茜素红染色法检测钙化结节。结果: 实时定量RT-PCR检测结果显示,与空白对照组比较,淫羊藿素组CXCR4SDF-1Runx2OPG基因表达量增加(P < 0.05或P < 0.01),而加入CXCR4拮抗剂AMD3100后,CXCR4基因相对表达量减少(P < 0.05)。蛋白质印迹法检测结果显示,与空白对照组比较,淫羊藿素组CXCR4、SDF-1、Runx2和OPG蛋白的相对表达量增加(均P < 0.01),而加入AMD3100后,CXCR4、SDF-1、Runx2和OPG蛋白表达量减少(均P < 0.01)。药物处理第3天和第6天时,淫羊藿素组ALP活性明显高于空白对照组(均P < 0.01),而加入AMD3100后ALP活性显著降低(均P < 0.01)。药物处理14 d时,与空白对照组比较,淫羊藿素组茜素红染色面积增加,而加入AMD3100后茜素红染色面积明显减少。结论: 淫羊藿素可能通过CXCR4/SDF-1信号通路促进小鼠成骨细胞成熟及矿化。


关键词: 成骨细胞/药物作用,  淫羊藿甙/药理学,  趋化因子, CXC/代谢,  受体, CXCR4/代谢,  细胞分化,  信号传导,  小鼠,  细胞, 培养的 
基因 引物序列(5′-3′) 长度(bp)
CXCR4 正向:AGTGACCCTCTGAGGCGTTTG
反向:GAAGCAGGGTTCCTTGTTGGAGT
1821
SDF-1 正向:GAGCCAACGTCAAACATCTGAA
反向:TCCAGGTACTCTTGGATCCACTTTA
1792
Runx2 正向:CCGCACGACAACCGCACCAT
反向:CGCTCCGGCCCACAACAAATCTC
5904
OPG 正向:CAGAGAAGCCACGCAAAAGTG
反向:AGCTGTGTCTCCGTTTTATCCT
1487
GAPDH 正向:AAATGGTGAAGGTCGGTGTG
反向:GAATTTGCCGTGAGTGGAGT
660
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Distribution of CXCR4 in MC3T3-E1 cells
Fig 2 mRNA expressions of CXCR4, SDF-1, Runx2 and OPG in each group
Fig 3 Electropherogram of CXCR4, SDF-1, OPG and Runx2 expression in each group
Fig 4 Relative expressions of CXCR4, SDF-1, Runx2 and OPG protein in each group
Fig 5 ALP activities in each group 3 d and 6 d after drug treatment
Fig 6 Comparison of stained area of calcified nodules in each group at 14 d of drug treatment
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