Damage accumulation in the bone under continuous daily loading causes local mechanical overloading known to induce osteocyte apoptosis, which promotes bone resorption to repair bone damage. However, only a few studies have investigated the mechanism of apoptosis in mechanically overloaded osteocytes. As mechanically stimulated osteocytes produce nitric oxide (NO), which triggers apoptosis in various cell types, we aimed to elucidate the mechanism underlying apoptosis in mechanically overloaded osteocytes, focusing on intracellular NO. To investigate the effects of force magnitude on apoptosis and intracellular NO production, we isolated osteocytes from DMP1-EGFP mice and subjected them to quantitative local forces via fibronectin-coated micro beads targeting integrin on the cell surface using a magnetic tweezer. Cell shrinkage was microscopically examined, and intracellular NO production was visualized using DAR-4 M. Mechanical stimulation revealed relationships between force magnitude, apoptosis, and intracellular NO production. The application of a smaller force resulted in no significant cell shrinkage or intracellular NO production; however, a larger force caused a rapid increase in intracellular NO production followed by cell shrinkage. Besides, intracellular NOS (NO synthase) inhibition and NO donation revealed the pro-apoptotic roles of NO in osteocytes. L-NAME (NOS inhibitor)-treated cells displayed no significant shrinkage under a larger force, whereas SNP (NO donor)-treated cells showed cell shrinkage and Annexin V fluorescence, indicating apoptosis. Collectively, our study demonstrates that larger force leads to NO production-mediated osteocyte shrinkage, implying an initial apoptotic response and highlighting the importance of NO production in bone damage.

在连续的每日负荷下，骨中的损伤累积会导致局部机械超负荷，从而导致骨细胞凋亡，从而促进骨吸收以修复骨损伤。然而，只有很少的研究调查了机械性超负荷骨细胞凋亡的机制。由于机械刺激的骨细胞产生一氧化氮（NO），从而触发各种细胞类型的细胞凋亡，因此我们旨在阐明细胞超负荷的机械性骨细胞中潜在的细胞凋亡机制。为了研究力的大小对细胞凋亡和细胞内NO产生的影响，我们从DMP1-EGFP小鼠中分离出骨细胞，并使用磁性镊子通过靶向细胞表面整联蛋白的纤连蛋白包被的微珠对它们施加定量的局部力。显微镜检查细胞的收缩，并使用DAR-4 M观察细胞内NO的产生。机械刺激揭示了力大小，凋亡和细胞内NO产生之间的关系。施加较小的力不会导致明显的细胞皱缩或细胞内NO产生。然而，更大的力导致细胞内NO产生迅速增加，随后细胞收缩。此外，细胞内NOS（NO合酶）抑制和NO捐赠揭示了NO在骨细胞中的促凋亡作用。经L-NAME（NOS抑制剂）处理的细胞在较大的作用力下无明显收缩，而经SNP（NO供体）处理的细胞显示细胞收缩和膜联蛋白V荧光，表明细胞凋亡。总的来说，