Swimming induces bone loss via regulating mechanical sensing pathways in bone marrow

Bone is an organ capable of perceiving external mechanical stress in real time and respond dynamically via mechanosensing proteins such as Piezo1 and YAP/TAZ. Upon sensing the mechano-signals, cells within the bone matrix collaborate to coordinate bone formation and resorption, while bone marrow cells are also stimulated and mobilized. High-load exercise stimulates osteoblast differentiation and bone formation. However, the mechanism through which the low-load exercises on bone homeostasis is still unclear. In this work, we established a long-term swimming training model to unload the mechanical stress in mice. Throughout the training model, we observed a significant loss in trabecular bone mass, as evidenced by microCT scanning and histological staining. Single-cell sequencing of the tibial bone marrow tissue revealed a significant increase in the percentage of bone marrow neutrophils, along with alterations in integrins and the ERK1/2 signaling pathway. Notably, the changes in both Integrins and the ERK1/2 signaling pathway in macrophages were more pronounced than in other cell types, which not only suggests a mechanical adaptive response in these cells. Moreover, the involvement of integrins is also critical for the crosstalk between monocyte precusors and macrophages during swimming. Together, this study provide a resource of the alterations of bone marrow cell gene expression profile during swimming and highlights the importance of Integrins and the ERK1/2 signaling pathway in the bone marrow microenvironment after swimming. Twelve 2-month-old male C57bl/6j mice, sourced from Shanghai Ninth People's Hospital, were housed in SPF conditions with free access to food and water, under a 12-hour light/dark cycle (07:00-19:00), at 22°C and 30-70% humidity. They were divided into swimming (n=6) and non-swimming (n=6) groups. The swimming group mice starting with 10 minutes of swimming on the first day and gradually increasing to 60 minutes per day within a week. Afterward, they swam for 60 minutes every day, five times a week, for a total of 12 weeks. All swimming training sessions were conducted in the afternoon. The non-swimming group mice did not undergo swimming training. After the previous training, three mice from the swimming group and three mice from the non-swimming group were euthanized. Bone marrow was flushed out from their tibiae using PBS, respectively. After dispersing the bone marrow tissue, dead cells were removed using a Dead Cell Removal Kit (130-090-101; Miltenyi Biotec) according to the manufacturer's instructions. Single-cell RNA sequencing (scRNA-seq) analysis of the tibial bone marrow cells was then performed using the 10x Genomics Chromium platform. We designed this experiment to explore the changes in bone marrow environment induced by mechanical stress unloading during swimming, and to provide an explanation for bone loss caused by swimming.

骨是一种能够实时感知外界机械应力，并通过Piezo1、YAP/TAZ等机械感应蛋白产生动态应答的器官。当感知到机械信号后，骨基质内的细胞会协同调控骨形成与骨吸收过程，同时骨髓细胞也会被激活并动员。大负荷运动可促进成骨细胞分化与骨形成，但低负荷运动对骨稳态的调控机制仍不明晰。本研究构建了小鼠长期游泳训练模型以实现机械应力卸载。通过microCT扫描与组织学染色证实，该训练模型可导致小鼠出现显著的骨小梁骨量丢失。对胫骨骨髓组织进行单细胞测序分析后发现，骨髓中性粒细胞占比显著升高，同时整合素（integrins）与ERK1/2信号通路发生改变。值得注意的是，巨噬细胞中整合素与ERK1/2信号通路的变化较其他细胞类型更为显著，这提示该类细胞存在机械适应性应答反应。此外，在游泳训练过程中，整合素的参与对单核前体细胞与巨噬细胞间的串扰亦至关重要。综上，本研究揭示了游泳训练过程中骨髓细胞基因表达谱的变化特征，同时阐明了整合素与ERK1/2信号通路在游泳后骨髓微环境中的重要作用。本研究选用12只2月龄雄性C57bl/6j小鼠，购自上海第九人民医院，饲养于SPF级环境中，自由进食饮水，光照周期为12小时明暗交替（07:00-19:00），环境温度维持在22℃，湿度控制在30%-70%。将小鼠随机分为游泳组（n=6）与对照组（非游泳组，n=6）。游泳组小鼠首日先进行10分钟游泳训练，于1周内逐步递增至每日60分钟；此后每日游泳60分钟，每周训练5次，持续共计12周，所有训练均于下午进行。对照组小鼠不接受游泳训练。训练12周后，分别处死游泳组与对照组各3只小鼠，采用PBS缓冲液冲洗胫骨以获取骨髓组织。将骨髓组织分散后，依照产品说明书使用死细胞去除试剂盒（Dead Cell Removal Kit，货号130-090-101；Miltenyi Biotec公司）去除死细胞。随后采用10x Genomics Chromium平台对胫骨骨髓细胞进行单细胞RNA测序（scRNA-seq）分析。本实验旨在探究游泳训练过程中机械应力卸载对骨髓微环境的影响，并为游泳诱导的骨丢失现象提供机制层面的解释。