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

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

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。

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-1 R/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-1 R 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-1 R/NO signaling pathway.