Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P < 0.01) after LIPUS stimulation. In addition, Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation, continued for 5 min, and then returned to their initial levels at 30 min. These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium. The influx of Ca²⁺ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.

机械强度刺激（例如低强度脉冲超声（LIPUS））已显示出促进骨再生和新鲜骨折修复的作用，但是涉及的基本生物物理机制仍然难以捉摸。在这里，我们建议Piezo1的机械敏感离子通道在无创超声诱导的机械传导途径中触发下游细胞信号过程起关键作用。本研究旨在研究LIPUS处理后Piezo1在MC3T3-E1细胞中的表达和作用。免疫荧光分析表明，Piezo1存在于MC3T3-E1细胞上，可通过shRNA转染消除。LIPUS刺激显着增加了MC3T3-E1细胞的迁移和增殖，而Piezo1的敲低限制了细胞迁移和增殖的增加。用Fluo-8标记后，与LIPUS刺激期间的基线相比，MC3T3-E1细胞显示出具有多个高峰的荧光强度曲线。在LIPUS刺激后，在shRNA-Piezo1细胞中未观察到荧光强度趋势的明显变化，这与GsMTx4处理组的结果相似。MC3T3-E1细胞中ERK1 / 2的磷酸化率显着增加（P  <0.01）LIPUS刺激后。此外，LIPUS刺激后，用鬼笔环肽-iFluor标记的F-肌动蛋白丝立即积聚在核周区域，持续5分钟，然后在30分钟时恢复到其初始水平。这些结果表明，Piezo1可以将LIPUS诱导的机械信号转化为细胞内钙。Ca ²⁺的流入充当第二信使，以激活ERK1 / 2磷酸化和核周F-肌动蛋白丝聚合，从而调节MC3T3-E1细胞的增殖。