Search JIM Advanced Search

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)
β-actin F: 5′-CCTGTACGCCAACACAGTGC-3′
R: 5′-ATACTCCTGCTTGCTGATCC-3′ 60 211
p38 F: 5′-CCAGATGCCGAAGATGAACT-3′
R: 5′-GGGCTGCTGTGATCCTCTTAT-3′ 60 113
p44 F: 5′-ATCAGCCCCTTTGAGCATC-3′
R: 5′-GTCTCCATGAGGTCCTGAACA-3′ 60 164
p42 F: 5′-TCCCAAATGCTGACTCCAAAG-3′
R: 5′-GCCAGAGCCTGTTCAACTTCA-3′ 60 91

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
[1] Fang-yuan Wang, Jian Jia, Huan-huan Song, Cheng-ming Jia, Chang-bo Chen, Jing Ma. Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress. Journal of Integrative Medicine, 2019, 17(3): 205-212.
[2] Syed Nasir Abbas Bukhari, Fahad Hussain, Hnin Ei Thu, Zahid Hussain. Synergistic effects of combined therapy of curcumin and Fructus Ligustri Lucidi for treatment of osteoporosis: cellular and molecular evidence of enhanced bone formation. Journal of Integrative Medicine, 2019, 17(1): 38-45.
[3] Uday P. Pratap, Hannah P. Priyanka, Karthik R. Ramanathan, Vishak Raman, Lalgi Hima, Srinivasan Thyagarajan. Noni (Morinda citrifolia L.) fruit juice delays immunosenescence in the lymphocytes in lymph nodes of old F344 rats. Journal of Integrative Medicine, 2018, 16(3): 199-207.
[4] Byung Hyuk Han, Yun Jung Lee, Jung Joo Yoon, Eun Sik Choi, Seung Namgung, Xian Jun Jin, Da Hye Jeong, Dae Gill Kang, Ho Sub Lee. Hwangryunhaedoktang exerts anti-inflammation on LPS-induced NO production by suppressing MAPK and NF-κB activation in RAW264.7 macrophages. Journal of Integrative Medicine, 2017, 15(4): 326-336.
[5] Shu Dong, Shi-bing Su​. Advances in mesenchymal stem cells combined with traditional Chinese medicine therapy for liver fibrosis. Journal of Integrative Medicine, 2014, 12(3): 147-155.
[6] Gui-lin Ouyang, Xiao-hui Feng, Lian-bo Xiao, Zheng Huang, Qing Xia, Da-peng Han, Fei Zhu . Effects of Chinese herbal medicine Qianggu Capsule on patients with rheumatoid arthritis-induced osteoporosis: a report of 82 cases. Journal of Chinese Integrative Medicine, 2012, 10(12): 1394-1399.
[7] Lei Liu, Yuan-hui Guo , Hai-liang Xin , Yan Nie , Ting Han, Lu-ping Qin , Oiao-yan Zhang. Antiosteoporotic effects of benzylbenzoate glucosides from Curculigo orchioides in ovariectomized rats. Journal of Chinese Integrative Medicine, 2012, 10(12): 1419-1426.
[8] Dong-tao Li, Jian Wang , Hong-yang Jiang , Feng-lei Shi , Fu-yu Li , Ji-hong Liu , Yong-mei Cheng , Nan Yan , Ai-hua Hu, Mei-zeng Zhang , Jie Li, Ling-bo Wei , Rong-qin Jiang. Quantitative evaluation of the degrees of traditional Chinese medicine qualitative syndromes of osteoporosis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1254-1262.
[9] Yuan-jie Wu , Zhao-hui Fang , Shu-guo Zheng , Yuan-bo Wu , Ai-hua Fan. Effects of Chinese herbal medicine Danzhi Jiangtang Capsule and exercise on JNK signaling pathway in pancreatic tissues of diabetic rats. Journal of Chinese Integrative Medicine, 2012, 10(11): 1279-1285.
[10] Lin-hua Qin, Jiao Lü, Lin Kong, Yun-xing Shi, Yong-ping Li, Guo-zhong Zhou, Zhi-wu Zheng, Lin Li, Xing-ying Ji. Andrographolide inhibits extracellular signal-regulated kinase 1/2 signaling pathway in activated macrophages. Journal of Chinese Integrative Medicine, 2011, 9(6): 632-637.
[11] Lei Bao, Shi-en Zou, Shao-fen Zhang. Dose-dependent effects of daidzein in regulating bone formation through estrogen receptors and peroxisome proliferator-activated receptor γ. Journal of Chinese Integrative Medicine, 2011, 9(2): 165-172.
[12] Zhi-ming Huang, Gui-ling Ouyang, Lian-bao Xiao, Ning-li Li, Hua-li Gao, Yong He, Zheng Huang, Xin-xing Huang. Effects of Drynaria total flavonoids on apoptosis of osteoblasts mediated by tumor necrosis factor-α. Journal of Chinese Integrative Medicine, 2011, 9(2): 173-178.
[13] Qin Bian, Jian-hua Huang, Zhu Yang, You Ning, Yong-jian Zhao, Yong-jun Wang, Zi-yin Shen. Effects of active ingredients in three kidney-tonifying Chinese herbal drugs on gene expression profile of bone marrow stromal cells from a rat model of corticosterone-induced osteoporosis. Journal of Chinese Integrative Medicine, 2011, 9(2): 179-185.
[14] Li Dong-tao, Li Fu-yu, Wang Jian, Liu Ji-hong, Yan Nan, Cheng Yong-mei, Hu Ai-hua, Jiang Hong-yang, Shi Feng-lei, Zhang Mei-zeng, Li Jie, Wei Ling-bo, Jiang Rong-qin. A study of diagnostic criteria for traditional Chinese medicine syndromes in osteoporosis. Journal of Chinese Integrative Medicine, 2011, 9(12): 1326-1332.
[15] Yang Chen, Jian-hua Huang, You Ning, Zi-yin Shen. Icariin and its pharmaceutical efficacy: Research progress of molecular mechanism. Journal of Chinese Integrative Medicine, 2011, 9(11): 1179-1184.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. An explanation on "guiding principles of clinical research on mild cognitive impairment (protocol)". Journal of Chinese Integrative Medicine, 2008, 6(1): 15-21
[4] Jin-rong Fu. Establishment of multivariate diagnosis and treatment system of modern gynecology of traditional Chinese medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 22-24
[5] Hao Li, Ming-jiang Yao, Wen-ming Zhao, Jie Guan, Lin-lin Cai, Ling Cui. A randomized, controlled, double-blind trial of Huannao Yicong capsule in senile patients with mild cognitive impairment. Journal of Chinese Integrative Medicine, 2008, 6(1): 25-31
[6] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[7] Yi-ting He, Qing-lin Zha, Jian-ping Yu, Yong Tan, Cheng Lu, Ai-ping Lv. Principal factor analysis of symptoms of rheumatoid arthritis and their correlations with efficacy of traditional Chinese medicine and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 32-36
[8] Jun Cai, Hua Wang, Sheng Zhou, Bin Wu, Hua-rong Song, Zheng-rong Xuan. Effect of Sijunzi Decoction and enteral nutrition on T-cell subsets and nutritional status in patients with gastric cancer after operation: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2008, 6(1): 37-40
[9] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[10] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50