ROLES OF SIGNAL MOLECULE p38 INVOLVED IN MINERALIZATION AND MATURATION OF OSTEOBLASTS PROMOTED BY LOW FREQUENCY PULSED ELECTROMAGNETIC FIELDS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FANGQingqing ¹ , LIZhizhong ² , ZHOUJian ¹ , YANJuanli ¹ , SHIWengui ¹ , XIEYanfang ¹ ,  CHENKeming ¹

1. Institute of Orthopaedics, Lanzhou General Hospital of Chinese PLA, Lanzhou Gansu, 730050, P. R. China;
2. College of Life Science and Engineering, Lanzhou University of Technology;

Corresponding author：

CHENKeming, Email: chkeming@yahoo.com

Keywords：

Osteoporosis; Pulsed electromagnetic fields; Osteoblast; Mitogen-activated protein kinase

DOI：

10.7507/1002-1892.20160253

Video：

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Objective To investigate whether signal molecule mitogen-activated protein kinases (MAPKs) involves in the process of the mineralization and maturation of rat calvarial osteoblasts promoted by 50 Hz, 0.6 mT pulsed electromagnetic fields. Methods Rat calvarial osteoblasts were obtained by enzyme digestion from the skull of 6 neonatal Wistar rats of SPF level. The primary osteoblasts were treated in 50 Hz and 0.6 mT pulsed electromagnetic fields for 0, 5, 10, 20, 40, 60, and 120 minutes; the protein expression of phosphorylated MAPKs was detected by Western blot. The osteoblasts were randomly divided into group A (control group), group B (low frequency pulse electromagnetic fields treatment group), group C (SB202190 group), and group D (SB202190+low frequency pulse electromagnetic fields treatment group); the alkaline phosphatase (ALP) activities were tested after corresponding treatment for 1, 4, and 7 days. The corresponding treated more than 90% confluenced osteoblasts were cultured under condition of osteogenic induction, then ALP staining and alizarin red staining were carried out at 9 and 12 days respectively. Results The results of Western blot showed that there was no significant changes in the protein expressions of phosphorylated level of extracellular signal-related kinases 1/2 and c-Jun amino N-terminal kinases 1/2 in 50 Hz, 0.6 mT pulsed electromagnetic fields P>0.05), but the phosphorylated level of p38 began to increase at 5 minutes, peaked at 40 minutes, then gradually decreased, and it was significantly higher at 5-120 minutes than at 0 minute (P<0.05). After the activities of p-p38 was inhibited by inhibitor SB202190, the ALP activities, positive colonies and area of ALP and calcified nodules of group B were significantly higher than groups A, C, and D (P<0.05). Conclusion p38 is one of the signal molecules involved in the process of the mineralization and maturation of rat calvarial osteoblasts promoted by 50 Hz, 0.6 mT pulsed electromagnetic fields.

Citation： FANG Qingqing, LI Zhizhong, ZHOU Jian, YAN Juanli, SHI Wengui, XIE Yanfang, CHEN Keming. ROLES OF SIGNAL MOLECULE p38 INVOLVED IN MINERALIZATION AND MATURATION OF OSTEOBLASTS PROMOTED BY LOW FREQUENCY PULSED ELECTROMAGNETIC FIELDS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1238-1243. doi: 10.7507/1002-1892.20160253 Copy

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2.	Hsieh TP, Sheu SY, Sun JS, et al. Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. Phytomedicine, 2011, 18(2-3):176-185.
3.	Hill PA. Bone remodelling. Bri J Orthod, 1998, 25(2):101-107.
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6.	Jing D, Cai J, Shen G, et al. The preventive effects of pulsed electromagnetic fields on diabetic bone loss in streptozotocin-treated rats. Osteoporos Int, 2010, 22(6):1885-1895.
7.	Tucker JJ, Cirone JM, Morris TR, et al. Pulsed electromagnetic field therapy improves tendon-to-bone healing in a rat rotator cuff repair model. J Orthop Res, 2016.[Epub ahead of print].
8.	Yang SH, Sharrocks AD, Whitmarsh AJ. MAP kinase signalling cascades and transcriptional regulation. Gene, 2013, 513(1):1-13.
9.	Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol, 1999, 11(2):211-218.
10.	Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol Mol Biol Rev, 2011, 75(1):50-83.
11.	Song L, Zhao J, Zhang X, et al. Icariin induces osteoblast proliferation, differentiation and mineralization through estrogen receptor-mediated ERK and JNK signal activation. Eur J Pharmacol, 2013, 714(1-3):15-22.
12.	Greenblatt MB, Shim JH, Zou W, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest, 2010, 120(7):2457-2473.
13.	Yan JL, Zhou J, Ma HP, et al. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia. Mol Cell Endocrinol, 2015, 404:132-140.
14.	翟中和, 王喜忠, 丁明孝. 细胞生物学. 2版. 北京:高等教育出版社, 2000:146-147.
15.	Li XD, Liu ZY, Chang B, et al. Panax notoginseng saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK and p38 MAPK signaling pathways. Cell Physiol Biochem, 2011, 28(2):367-376.
16.	Xiao HH, Gao QG, Zhang Y, et al. Vanillic acid exerts oestrogen-like activities in osteoblast-like UMR 106 cells through MAP kinase (MEK/ERK)-mediated ER signaling pathway. J Steroid Biochem Mol Biol, 2014, 144 Pt B:382-391.
17.	Guicheux J, Lemonnier J, Ghayor C, et al. Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. J Bone Miner Res, 2003, 18(11):2060-2068.
18.	Groah SL, Lichy AM, Libin AV, et al. Intensive electrical stimulation attenuates femoral bone loss in acute spinal cord injury. PM R, 2010, 2(12):1080-1087.
19.	Griffin XL, Costa ML, Parsons N, et al. Electromagnetic field stimulation for treating delayed union or non-union of long bone fractures in adults. Cochrane Database Syst Rev, 2011, (4):CD008471.
20.	Chalidis B, Sachinis N, Assiotis A, et al. Stimulation of bone formation and fracture healing with pulsed electromagnetic fields:biologic responses and clinical implications. Int J Immunopathol Pharmacol, 2011, 24(1 Suppl 2):17-20.
21.	Pilla A, Fitzsimmons R, Muehsam D, et al. Electromagnetic fields as first messenger in biological signaling:Application to calmodulin-dependent signaling in tissue repair. Biochim Biophys Acta, 2011, 1810(12):1236-1245.
22.	Pall ML. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects. J Cell Mol Med, 2013, 17(8):958-965.
23.	Jing D, Li F, Jiang M, et al. Pulsed electromagnetic fields improve bone microstructure and strength in ovariectomized rats through a Wnt/Lrp5/β-catenin signaling-associated mechanism. PLoS One, 2013, 8(11):e79377.

1. Kular J, Tickner J, Chim SM, et al. An overview of the regulation of bone remodelling at the cellular level. Clin Biochem, 2012, 45(12):863-873.
2. Hsieh TP, Sheu SY, Sun JS, et al. Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. Phytomedicine, 2011, 18(2-3):176-185.
3. Hill PA. Bone remodelling. Bri J Orthod, 1998, 25(2):101-107.
4. Kanis JA, Borgstrom F, De Laet C, et al. Assessment of fracture risk. Osteoporos Int, 2005, 16(6):581-589.
5. Tsai MT, Li WJ, Tuan RS, et al. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res, 2009, 27(9):1169-1174.
6. Jing D, Cai J, Shen G, et al. The preventive effects of pulsed electromagnetic fields on diabetic bone loss in streptozotocin-treated rats. Osteoporos Int, 2010, 22(6):1885-1895.
7. Tucker JJ, Cirone JM, Morris TR, et al. Pulsed electromagnetic field therapy improves tendon-to-bone healing in a rat rotator cuff repair model. J Orthop Res, 2016.[Epub ahead of print].
8. Yang SH, Sharrocks AD, Whitmarsh AJ. MAP kinase signalling cascades and transcriptional regulation. Gene, 2013, 513(1):1-13.
9. Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol, 1999, 11(2):211-218.
10. Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol Mol Biol Rev, 2011, 75(1):50-83.
11. Song L, Zhao J, Zhang X, et al. Icariin induces osteoblast proliferation, differentiation and mineralization through estrogen receptor-mediated ERK and JNK signal activation. Eur J Pharmacol, 2013, 714(1-3):15-22.
12. Greenblatt MB, Shim JH, Zou W, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest, 2010, 120(7):2457-2473.
13. Yan JL, Zhou J, Ma HP, et al. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia. Mol Cell Endocrinol, 2015, 404:132-140.
14. 翟中和, 王喜忠, 丁明孝. 细胞生物学. 2版. 北京:高等教育出版社, 2000:146-147.
15. Li XD, Liu ZY, Chang B, et al. Panax notoginseng saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK and p38 MAPK signaling pathways. Cell Physiol Biochem, 2011, 28(2):367-376.
16. Xiao HH, Gao QG, Zhang Y, et al. Vanillic acid exerts oestrogen-like activities in osteoblast-like UMR 106 cells through MAP kinase (MEK/ERK)-mediated ER signaling pathway. J Steroid Biochem Mol Biol, 2014, 144 Pt B:382-391.
17. Guicheux J, Lemonnier J, Ghayor C, et al. Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. J Bone Miner Res, 2003, 18(11):2060-2068.
18. Groah SL, Lichy AM, Libin AV, et al. Intensive electrical stimulation attenuates femoral bone loss in acute spinal cord injury. PM R, 2010, 2(12):1080-1087.
19. Griffin XL, Costa ML, Parsons N, et al. Electromagnetic field stimulation for treating delayed union or non-union of long bone fractures in adults. Cochrane Database Syst Rev, 2011, (4):CD008471.
20. Chalidis B, Sachinis N, Assiotis A, et al. Stimulation of bone formation and fracture healing with pulsed electromagnetic fields:biologic responses and clinical implications. Int J Immunopathol Pharmacol, 2011, 24(1 Suppl 2):17-20.
21. Pilla A, Fitzsimmons R, Muehsam D, et al. Electromagnetic fields as first messenger in biological signaling:Application to calmodulin-dependent signaling in tissue repair. Biochim Biophys Acta, 2011, 1810(12):1236-1245.
22. Pall ML. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects. J Cell Mol Med, 2013, 17(8):958-965.
23. Jing D, Li F, Jiang M, et al. Pulsed electromagnetic fields improve bone microstructure and strength in ovariectomized rats through a Wnt/Lrp5/β-catenin signaling-associated mechanism. PLoS One, 2013, 8(11):e79377.

Chinese Journal of Reparative and Reconstructive Surgery

ROLES OF SIGNAL MOLECULE p38 INVOLVED IN MINERALIZATION AND MATURATION OF OSTEOBLASTS PROMOTED BY LOW FREQUENCY PULSED ELECTROMAGNETIC FIELDS

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