Effect of low-frequency pulsed electromagnetic fields on activity of rat calvarial osteoblasts through IGF-1R/NO signaling pathway

doi:10.3785/j.issn.1008-9292.2019.04.06

J Zhejiang Univ (Med Sci)

2019, Vol. 48

Issue (2): 158-164 DOI: 10.3785/j.issn.1008-9292.2019.04.06

Effect of low-frequency pulsed electromagnetic fields on activity of rat calvarial osteoblasts through IGF-1R/NO signaling pathway

SHAO Jiale^1,²(

),LI Zhizhong²,ZHOU Jian¹,LI Kai¹,QIN Rong¹,CHEN Keming^1,*(

)

1. Institute of Orthopedic Research, the 940^th Hospital of Joint Logistics Support Force of People's Liberation Army, Lanzhou 730050, China
2. School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China

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Abstract

Objective: To investigate the effect of low-frequency pulsed electromagnetic fields (PEMF) on the maturation and mineralization of rat cranial osteoblasts in vitro and its relation to IGF-1R/NO signaling pathway. Methods: The rat osteoblasts were isolated and cultured in vitro and randomly divided into blank control group, PEMF group, GSK group (IGF-1R blocker) and PEMF+GSK group. The cells were treated with 50 Hz 0.6 mT PEMF for 1.5 h/d. After 3 d of PEMF treatment, the expressions of protein kinase (AKT), inducible nitric oxide synthase (iNOS) and cGMP-dependent protein kinase (PKG) were detected by Western blotting; on 6 d of PEMF treatment alkaline phosphatase (ALP) activity was determined; on 12 d of PEMF treatment the calcification nodule formation was demonstrated by Alizarin red staining. Results: NO level was significantly increased in rat osteoblasts treated with 50 Hz 0.6 mT PEMF for 1.5 h/d. Western blot analysis showed that the expressions of AKT, iNOS and PKG protein in PEMF group were higher than those in the control group (all P < 0.01); the ALP activity was increased(P < 0.05), and the PEMF group had the largest area of Alizarin red staining (P < 0.01). The expressions of AKT, iNOS and PKG protein in GSK group were lower than those in the control group; the ALP activity was decreased (P < 0.05), and the GSK group had the least area of Alizarin red staining (P < 0.01). The expressions of AKT, iNOS, PKG protein, the ALP activity and the area of Alizarin red staining in PEMF+GSK group were between PEMF group and GSK group. Conclusion: PEMF may enhance the maturation and mineralization of rat cranial osteoblasts in vitro through IGF-1R/NO signaling pathway.

Key words： Osteoblasts/radiation effects Skull/cytology Electromagnetic fields Glycogen synthase kinase 3/biosynthesis Receptor, IGF type 1/biosynthesis Signal transduction Cells, cultured Rats, Wistar

Received: 15 October 2018 Published: 24 July 2019

CLC:	Q683
	Q291
	R68

Corresponding Authors: CHEN Keming E-mail: shaojiale1119@163.com;chenkm@lut.cn

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	SHAO Jiale
	LI Zhizhong
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	LI Kai
	QIN Rong
	CHEN Keming

Cite this article:

SHAO Jiale,LI Zhizhong,ZHOU Jian,LI Kai,QIN Rong,CHEN Keming. Effect of low-frequency pulsed electromagnetic fields on activity of rat calvarial osteoblasts through IGF-1R/NO signaling pathway. J Zhejiang Univ (Med Sci), 2019, 48(2): 158-164.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2019.04.06 OR http://www.zjujournals.com/med/Y2019/V48/I2/158

低频脉冲电磁场通过IGF-1R/NO信号通路促进大鼠颅骨成骨细胞成熟及矿化

目的: 观察低频脉冲电磁场（PEMF）是否通过胰岛素样生长因子1受体（IGF-1R）/一氧化氮（NO）信号通路促进大鼠颅骨成骨细胞成熟及矿化。方法: 体外分离培养乳鼠颅骨成骨细胞，传代后随机分成9组，每天用50 Hz 0.6 mT PEMF处理不同的时间，通过检测成骨细胞中NO含量确定PEMF最佳处理时长。将传代后的成骨细胞随机分成空白对照组、PEMF组、GSK（IGF-1R阻断剂）组、PEMF+GSK组，PEMF处理成骨细胞3 d后，采用蛋白质印迹法检测蛋白激酶（AKT）、诱导型一氧化氮合酶（iNOS）和cGMP依赖性蛋白激酶（PKG）蛋白表达水平；PEMF处理第6天时测定碱性磷酸酶（ALP）活性；PEMF处理第12天时采用茜红素染色法观察钙化结节形成情况。结果: 经1.5 h电磁场处理后的成骨细胞中NO含量显著提高（P < 0.01），遂后续采用PEMF处理1.5 h。与空白对照组比较，PEMF组AKT、iNOS、PKG蛋白表达量增加（均P < 0.01），ALP活性升高（P < 0.05），且茜红素染色面积增大（P < 0.01）；GSK组AKT、iNOS、PKG蛋白表达量减少，ALP活性降低（P < 0.05），茜红素染色面积最小（P < 0.01）；PEMF+GSK组上述实验结果均高于GSK组但低于PEMF组（P < 0.05或P < 0.01）。结论: PEMF可能通过IGF-1R/NO信号通路促进大鼠颅骨成骨细胞成熟与矿化。

关键词： 成骨细胞/辐射效应, 颅骨/细胞学, 电磁场, 糖原合成酶激酶3/生物合成, 受体, IGF1型/生物合成, 信号传导, 细胞, 培养的, 大鼠, Wistar

Fig 1 Expression of nitric oxide in rat calvarial osteoblasts treated with pulsed electromagnetic filed

Fig 2 The AKT, iNOS and PKG protein expression in rat calvarial osteoblasts treated with pulsed electromagnetic field

Fig 3 Alizarin red staining showed calcium nodule in rat calvarial osteoblasts after 12 days of pulsed electromagnetic field treatment


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