低强度脉冲超声通过PIEZO1/PI3K/AKT/mTOR信号通路促进成骨细胞增殖的研究

Study of promotion of low-intensity pulsed ultrasound for osteoblast proliferation through PIEZO1/PI3K/AKT/mTOR signaling pathway

目的探索低强度脉冲超声(low-intensity pulsed ultrasound, LIPUS)的促成骨机制,为治疗骨质疏松寻找潜在有价值的分子靶点。方法将成骨细胞分为LIPUS组和对照组,分别给予LIPUS 1、6、12、18、24、36 h照射后,使用CCK-8检测两组细胞的增殖活力,并使用Western blot检测两组PIEZO1蛋白的表达水平。将成骨细胞分为对照组、LIPUS组、LIPUS+GsMTx4组和GsMTx4组,给予干预措施后,使用Western blot检测各组p-PI3K、PI3K、p-AKT、AKT、p-mTOR、mTOR和Cyclin D1的表达水平。结果 CCK-8结果提示,LIPUS可显著促进MC3T3-E1成骨细胞的增殖活力;LIPUS组的PIEZO1和Cyclin D1的表达水平显著高于对照组(P<0.001),表明LIPUS可能是通过上调PIEZO1的表达进而促进MC3T3-E1成骨细胞的增殖;在机制研究中发现,LIPUS组p-PI3K、p-AKT、p-mTOR和Cyclin D1的表达水平显著高于对照组(P<0.001),GsMTx4组的显著低于对照组(P<0.001),LIPUS+GsMTx4组的显著低于LIPUS组(P<0.001)。结论 LIPUS可通过促进PIEZO1的表达进而活化PI3K/AKT/mTOR信号通路促进MC3T3-E1成骨细胞的增殖。

Objective To explore the mechanism of low-intensity pulsed ultrasound(LIPUS) in promoting bone formation, and to find potential valuable molecular targets for the treatment of osteoporosis. Methods Osteoblasts were divided into LIPUS group and Control group. After being irradiated with LIPUS for 1 h, 6 h, 12 h, 18 h, 24 h, and 36 h, the cell proliferation viability of the two groups was detected with CCK-8 assay. The expression level of PIEZO1 protein in the two groups was detected with Western blotting. Then the osteoblasts were divided into Control group, LIPUS group, LIPUS+GsMTx4 group, and GsMTx4 group. After intervention, Western blotting was used to detect the expression levels of p-PI3 K, PI3 K, p-AKT, AKT, p-mTOR, mTOR, and Cyclin D1 in each group. Results CCK-8 result suggested that LIPUS significantly promoted the proliferation of MC3 T3-E1 osteoblasts. The expression levels of PIEZO1 and Cyclin D1 in the LIPUS group were significantly higher than those in the Control group(P<0.001), which indicated that LIPUS promoted the proliferation of MC3 T3-E1 osteoblasts by up-regulating the expression of PIEZO1. In the mechanism assay, it was found that the expression levels of p-PI3 K, p-AKT, p-mTOR, and Cyclin D1 in the LIPUS group were significantly higher than those in the Control group(P<0.001). The expression levels of p-PI3 K, p-AKT, p-mTOR, and Cyclin D1 were significantly lower in the GsMTx4 group than those in the Control group(P<0.001), and were significantly lower in the LIPUS+GsMTx4 group than in the LIPUS group(P<0.001). Conclusion LIPUS promotes the proliferation of MC3 T3-E1 osteoblasts by promoting the expression of PIEZO1 to activate the PI3 K/AKT/mTOR signaling pathway.