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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (11): 1272-1278.doi: 10.3736/jcim20121111

• Original Experimental Research • Previous Articles     Next Articles

Analysis of the osteogenetic effects exerted on mesenchymal stem cell strain C3H10T1/2 by icariin via MAPK signaling pathway in vitro

Xiang-ying 1,2, Qin Bian1,3(), Zi-yin Shen1   

  1. 1. Institute of Chinese Integrative Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
    2. School of Life Science, Fudan University, Shanghai 200433, China
    3. Institute of Spine Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Received:2012-02-01 Accepted:2012-03-30 Online:2012-11-20 Published:2018-11-15

Objective: To investigate the effects of icariin, an effective extract from traditional Chinese medicine Epimedium pubescens with the function of tonifying kidney, in promoting osteogenesis of mesenchymal stem cell line C3H10T1/2, and to explore the underlying mechanism.

Methods: After culture with icariin (0, 10 -7, 10 -6, 10 -5, and 10 -4 mol/L) and osteogenic supplement for 26 d in vitro, osteogenic differentiation of C3H10T1/2 cells was detected by alkaline phosphatase (ALP) assay. The RNA was extracted from cells cultured with 10 -5 mol/L icariin for 2, 8, 24 and 48 hours, and mRNA expressions of p38, p42 and p44 were measured using real-time reverse transcription-polymerase chain reaction (PCR) method. Three main proteins of MAPK signaling pathway (p38, and extracellular signal-regulated protein kinase (ERK), also named p42/44) and c-Jun N-terminal kinase (JNK) and their phospho-products were examined using Western blotting after icariin treatments of 10, 30, 60 and 120 min.

Rethods: Icariin at a dose of 10 -5 mol/L, when combined with the osteogenic supplement, had the best ability to promote osteogenic differentiation on C3H10T1/2 cells. Based on real-time PCR, the authors found that after two-hour ICA treatment, the gene expression of p38 revealed a significant decline compared with the control group (P<0.01). The levels of p42 and p44 mRNAs were decreased greatly after two-hour ICA treatment, while increased after 48-hour ICA treatment (P<0.05, P<0.01). There was no significant difference at other time points (P>0.05). Phospho-p42 was decreased after 10-minute icariin treatment, while phospho-p38 expression displayed an increase after 10- and 30-minute of treatment with icariin. There was no notable difference in phospho-JNK expression at these four time points.

Conclusion: Icariin promotes differentiation of the mesenchymal stem cells C3H10T1/2 into osteoblasts, and its effect is related to the restraining of ERK expression and activation of p38 expression in the MAPK signaling pathway.

Key words: icariin, mitogen-activated protein kinase kinase, p38 mitogen-activated protein kinase, mitogen-activated protein kinase, mesenchymal stem cells, osteoporosis

Table 1

Information of antibodies and their dilution"

Antibody Company Species Dilution
p38 Cell signaling Rabbit polyclonal 1∶1 000
p-p38 Cell signaling Rabbit polyclonal 1∶1 000
p42/44 Cell signaling Rabbit polyclonal 1∶1 000
p-p42/44 Cell signaling Rabbit polyclonal 1∶1 000
JNK Cell signaling Rabbit polyclonal 1∶1 000
p-JNK Cell signaling Rabbit polyclonal 1∶1 000
PKCα Sigma Rabbit polyclonal 1∶1 000
β-actin Sigma Mouse monoclonal 1∶4 000
Anti-mouse IgG Sigma Goat polyclonal 1∶4 000
Anti-rabbit IgG Sigma Goat polyclonal 1∶8 000

Table 2

Primers of real-time fluorescent quantitative polymerase chain reaction"

Gene Primer sequence Annealing temperature (℃) Product size (bp)

Figure 1

Results of alkaline phosphatase staining"

Table 3

Results of p38, p42 and p44 mRNA expression (x±s)"

Group n p38 p42 p44
Quantify P value Quantify P value Quantify P value
2 h control 3 0.013 3±0.001 4 0.009 6 0.063 5±0.003 5 0.000 9 0.048 7±0.006 4 0.009 9
2 h icariin 3 0.008 3±0.001 2 0.039 3±0.003 3 0.028 6±0.004 0
8 h control 3 0.015 7±0.001 2 0.859 1 0.054 3±0.003 7 0.677 9 0.057 8±0.008 3 0.070 3
8 h icariin 3 0.015 9±0.001 8 0.052 6±0.005 5 0.046 0±0.000 8
24 h control 3 0.014 3±0.000 8 0.272 4 0.041 0±0.001 4 0.287 6 0.051 9±0.004 0 0.144 1
24 h icariin 3 0.016 6±0.003 0 0.047 1±0.008 5 0.060 8±0.007 4
48 h control 3 0.010 2±0.000 4 0.301 7 0.030 0±0.002 7 0.037 0 0.035 2±0.004 7 0.009 8
48 h icariin 3 0.011 5±0.001 8 0.040 5±0.005 3 0.052 1±0.004 2

Figure 2

Effects of icariin on the activation of p38, p42 and JNK tested by Western blotting DMSO: dimethyl sulfoxide; ICA: icariin; p-p38: phospho-p38; p-p42: phospho-p42; p-JNK: phospho-c-Jun N-terminal kinase; JNK: c-Jun N-terminal kinase."

[1] Bian Q, Liu SF, Huang JH, Yang Z, Ning Y, Zhao YJ, Wang YJ, Shen ZY. Regulation and control of three active components of TCM effective in reinforcing kidney on ovariotomy-induced osteoporosis rat 5s bone marrow stem cells revealed by gene microarray. Zhonghua Zhong Yi Yao Za Zhi. 2011; 26(5) : 889-893.
卞琴, 刘书芬, 黄建华, 杨铸, 宁友, 赵永见, 王拥军, 沈自尹 . 3种补肾中药有效成分对去卵巢骨质疏松大鼠骨髓间充质干细胞的调控作用.中华中医药杂志. 2011; 26(5) : 889-893.
[2] Bao JR, Yang JW, Li SF, Zhao W, Zhang QL, Yan YQ. Effects of icariin on ovariectomized osteoporotic rats. Wei Sheng Yan Jiu. 2005; 34(2) : 191-193. Chinese with abstract in English.
doi: 10.3969/j.issn.1000-8020.2005.02.021
鲍加荣, 杨继文, 李树峰, 赵崴, 张清兰, 严云勤 . 淫羊藿苷对去卵巢大鼠骨质疏松症的影响.卫生研究. 2005; 34(2) : 191-193.
doi: 10.3969/j.issn.1000-8020.2005.02.021
[3] Nian H, Ma MH, Nian SS, Xu LL . Antiosteoporotic activity of icariin in ovariectomized rats. Phytomedicine. 2009; 16(4) : 320-326.
doi: 10.1016/j.phymed.2008.12.006 pmid: 19269147
[4] Xiao Q, Chen A, Guo F . Effects of icariin on expression of OPN mRNA and type I collagen in rat osteoblasts in vitro.[J] Huazhong Univ Sci Technolog Med Sci. 2005; 25(6) : 690-692.
doi: 10.1007/BF02896172
[5] Chen KM, Ma HP, Ge BF, Liu XY, Ma LP, Bai MH, Wang Y . Icariin enhances the osteogenic differentiation of bone marrow stromal cells but has no effects on the differentiation of newborn calvarial osteoblasts of rats. Pharmazie. 2007; 62(10):785-789.
[6] Hsieh TP, Sheu SY, Sun JS, Chen MH, Liu MH . Icariin isolated from Epimedium pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfal expression. Phytomedicine. 2010; 17(6):414-423.
doi: 10.1016/j.phymed.2009.08.007
[7] Liu HJ, Wang XP, Lin J, Yu YC, Jiang XQ, Zhang XL, Zhou ZT, Zhang WD. The effect of icariin and astragalosid I on the proliferation and differentiation of bone marrow stromal cells. Zhong Yao Cai. 2006; 29(10):1062-1065. Chinese with abstract inEnglish.
刘海江, 王小平, 林娟, 余优成, 蒋欣泉, 张秀丽, 周曾同, 张卫东 . 淫羊藿苷和黄芪苷I对骨髓基质细胞增殖及成骨能力的影响.中药材. 2006; 29(10):1062-1065.
[8] Yin XX, Chen ZQ, Dang GT, Ma QJ, Liu ZJ. Effects of Epimedium pubescens icariine on proliferation and differentiation of human osteoblasts. Zhongguo Zhong Yao Za Zhi. 2005 ; 30(4) : 289-291. Chinese with abstract in English.
doi: 10.3321/j.issn:1001-5302.2005.04.016
殷晓雪, 陈仲强, 党耕町, 马庆军, 刘忠军 . 淫羊藿苷对人成骨细胞增殖与分化的影响.中国中药杂志. 2005; 30(4) : 289-291.
doi: 10.3321/j.issn:1001-5302.2005.04.016
[9] Chen KM, Ge BF, Ma HP, Liu XY, Bai MH, Wang Y . Icariin, a flavonoid from the herb Epimedium enhances the osteogenic differentiation of rat primary bone marrow stromal cells. Pharmazie. 2005; 60(12):939-942.
[10] Yin XX, Chen ZQ, Liu ZJ, Ma QJ, Dang GT . Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2. Chin Med J (Engl). 2007; 120(3):204-210.
[11] Zhao J, Ohba S, Shinkai M, Chung UI, Nagamune T . Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun. 2008; 369(2):444-448.
doi: 10.1016/j.bbrc.2008.02.054 pmid: 18295595
[12] Zhang G, Qin L, Shi Y . Epi?nediumr derived phytoestrogen flavonoids exert beneficial effect on preventing bone loss in late postmenopausal women: a 24-month randomized, double-blind and placebo-controlled trial.[J] Bone Miner Res. 2007; 22(7) : 1072-1079.
doi: 10.1359/jbmr.070405
[13] Li J, Zhao Z, Liu J, Huang N, Long D, Wang J, Li X, Liu Y . MEK/ERK and p38 MAPK regulate chon- drogenesis of rat bone marrow mesenchymal stem cells through delicate interaction with TGF-pi/Smads pathway. Cell Prolif. 2010; 43(4):333-343.
doi: 10.1111/j.1365-2184.2010.00682.x
[14] Jadlowiec J, Koch H, Zhang X, Campbell PG, Seyedain M, Sfeir C . Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway.[J] Biol Chem. 2004; 279(51):53323-53330.
doi: 10.1074/jbc.M404934200
[15] Higuchi C, Myoui A, Hashimoto N, Kuriyama K, Yoshioka K, Yoshikawa H, Itoh K . Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix.[J] Bone Miner Res. 2002; 17(10):1785-1794.
doi: 10.1359/jbmr.2002.17.10.1785
[16] Li JJ, Yu SF, Li TJ, Pang SZ. In vitro study of the effects of Epimedium on osteoclastic bone resorption in various oral mineralized tissues. Zhonghua Kou Qiang Yi Xue Za Zhi. 2002; 37(5):391-394. Chinese with abstract in English.
doi: 10.3760/j.issn:1002-0098.2002.05.023
李晶晶, 于世凤, 李铁军, 庞淑珍 . 淫羊藿对口腔各矿化组织破骨细胞性骨吸收的体外实验研究.中华口腔医学杂志. 2002; 37(5):391-394.
doi: 10.3760/j.issn:1002-0098.2002.05.023
[17] Meng FH, Li YB, Xiong ZL, Jiang ZM, Li FM . Osteoblastic proliferative activity of Y.pimedium brevicomum Maxim. Phytomedicine. 2005; 12(3):189-193.
doi: 10.1016/j.phymed.2004.03.007 pmid: 15830840
[18] Luo Y, Liu Y, Zhang Q. Effects of MAPK signaling pathway in osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells. Jilin Yi Xue. 2008; 29(6):443-445.Chinese.
罗杨, 刘一, 张科强 . MAPK信号传导通路在间充质干细胞向成骨细胞分化中的作用.吉林医学. 2008; 29(6):443-445.
[19] Zhou H, Yang X, Wang N, Zhang Y, Cai G . Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells. Mol Cell Endocrinol. 2007; 270(1-2):17-22.
doi: 10.1016/j.mce.2007.01.017
[20] Ding L, Liang XG, Hu Y, Zhu DY, Lou YJ . Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro. Stem Cells Dev. 2008; 17(4):751-760.
doi: 10.1089/scd.2007.0206
[21] Liao QC, Xiao ZS, Qin YF, Zhao Y, Pan W, Liu T, Zhou HH. p44/42 and p38 MAPKs exert differenct functions in osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cell cultures.Zhongguo Gu Zhi Shu Song Za Zhi. 2004; 10(3) : 267-271. Chinese with abstract in English.
廖清船, 肖洲生, 秦艳芳, 赵彦, 潘讳, 刘霆, 周宏灏 . P44/42和p38 MAPKs在骨髓间充质干细胞向成骨细胞分化中发挥不同的功能.中国骨质疏松杂志. 2004; 10(3) : 267-271.
[22] Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF . Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.[J] Bio Clmm. 2000; 275(13):9645-9652
[23] Lee DY, Cheng CK, Chiu JJ. Study of gene expression in osteoblasts induced shear stress. Zhonghua Chuang Shang Gu Ke Za Zhi. 2006; 8(10):911-913. Chinese with abstract in English.
doi: 10.3760/cma.j.issn.1671-7600.2006.10.004
李定宇, 郑诚功, 裘正健 . 剪切应力诱导成骨相关基因表达的研究.中华创伤骨科杂志. 2006; 8(10):911-913.
doi: 10.3760/cma.j.issn.1671-7600.2006.10.004
[24] Arbabi S, Maier RV . Mitogen-activated protein kinases. Crit Care Med. 2002; 30(1 Suppl) : S74-S79.
doi: 10.1097/00003246-200201001-00010
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