基于YAP与Wnt/β-catenin信号通路crosstalk探讨低强度脉冲超声治疗绝经后骨质疏松症的作用机制

Postmenopausal osteoporosis harms the physical and mental health of elderly women seriously. It is a current research hotspot in treating osteoporosis safely and effectively, which also meets the clinical needs. Recently, the role of low-intensity pulsed ultrasound in bone regeneration has gained increasing attention while research on LIPUS as a treatment for osteoporosis is still in its infancy. We demonstrated that LIPUS can promote bone regeneration by regulating subchondral osteogenesis（Bone,2018,110:47-57）. Previous studies showed that LIPUS can significantly improve bone mineral density in ovariectomized mice. However, the target and mechanism through which LIPUS promotes BMD remains unknown. YAP is a key molecule whereby cells sense external stress and convert it into biological signals. It has been reported that YAP can combine with β-catenin as a complex and initiate downstream biological reactions. We reported that LIPUS significantly improves YAP activity and activates the Wnt/β-catenin signaling pathway. Thus, we posit that LIPUS improves osteoporosis by regulating crosstalk between YAP and Wnt/β-catenin signaling pathways. It is believed that our project will provide a theoretical basis for the transformation of LIPUS into a treatment for osteoporosis.

绝经后骨质疏松症严重危害老年女性健康，如何安全有效治疗骨质疏松是目前临床需求和研究热点。近年来，低强度脉冲超声（LIPUS）在骨再生过程中的作用受到广泛关注，但LIPUS治疗骨质疏松的相关研究目前尚处于起步阶段。申请人已报道LIPUS可通过调控软骨下成骨促进骨再生（Bone,2018,110:47-57），前期研究证实LIPUS能够显著提高去卵巢小鼠骨密度，但LIPUS治疗骨质疏松的靶点和机制目前未知。申请人通过高通量测序发现LIPUS可促进YAP基因表达，同时激活Wnt/β-catenin信号通路。YAP是细胞感受外界应力并转导为生物学信号的关键分子，同时YAP与β-catenin可形成复合体启动下游生物学效应。据此申请人提出假说：LIPUS通过调控YAP与Wnt/β-catenin信号通路之间crosstalk改善骨质疏松，该课题可为LIPUS临床转化用于治疗骨质疏松提供确的理论依据。

绝经后骨质疏松严重影响老年女性生活质量，已有研究证实低强度脉冲超声（LIPUS）可以促进骨再生，本项目从整体，组织，细胞，分子四个层面证实了LIPUS可以改善绝经后骨质疏松。通过microct发现，LIPUS作用四周后，小鼠骨密度体现出显著差异（0.031 vs 0.042），相对骨体积显著增高（对照组2.75，LIPUS组4.02），骨小梁数量和骨小梁厚度同样显著提高（Tb.N 0.59 vs 0.77， Tb.Th 0.051 vs 0.072）。组织水平上，通过HE染色观察发现，在LIPUS作用下，骨小梁体积显著上升，提示LIPUS可以改善绝经后骨质疏松。通过茜素红和ALP染色证实LIPUS的促成骨效应。细胞实验中，对BMSC进行LIPUS干预后，发现其成骨相关基因和蛋白均显著升高，此外，通过高通量测序发现，Wnt信号通路在这一过程中起关键作用。由于Yap1与细胞核定位有密切关系，因此我们通过免疫荧光发现LIPUS干预显著增加了Yap1的入核率，通过抑制和下调实验，我们验证了Yap与β-catenin结合后共同入核发挥生物学效应，从而阐明了LIPUS改善绝经后骨质疏松的内在机制。