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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

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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."

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