基于维生素D促进肌纤维形成改善肌肉衰减综合征作用机制的研究

Human and rodent aging is characterized by the progressive decline of skeletal muscle mass and strength, a condition known as sarcopenia. Skeletal muscle arises from the fusion of muscle stem cells (also known as muscle satellite cells)/precursor myoblasts into multinucleated myofibres. One mechanism of sarcopenia is the decrease of both myofiber numbers and size by functional decline in muscle stem cells/precursor myoblasts. Vitamin D deficiency/insufficiency is prevalent in the Chinese population in almost all age groups and areas. Moreover, intracellular vitamin D receptor (VDR) is expressed in human skeletal muscle tissue. To date, several experimental studies have demonstrated the effects of active vitamin D and identify their potential mechanisms of action on myoblast proliferation and differentiation. However, the myoblast fusion-improved properties and mechanisms of vitamin D are not as well understood. We found that serum 25-(OH)D levels were closely related to the prevalence of sarcopenia among the elderly population. Moreover, the experimental results suggest that active vitamin D treatment has improved the fusion of precursor myoblasts into multinucleated myofibres. Since muscle stem cells/precursor myoblasts expressed VDR at mRNA and corresponding protein levels, and the active Brain-specific Angiogenesis Inhibitor-1 (BAI1)-mediated ELMO/Dock180/Rac1 signaling pathway provides a crucial mechanism for fusion of myoblasts, we hypothesized that vitamin D is a functional component effective towards prevention of sarcopenia, and that this effect might involve the BAI1-mediated ELMO/Dock180/Rac1 signaling pathway. The primary objective of the present research is to examine the effects of vitamin D on the progression of sarcopenia and the myoblast fusion in an animal model of aging in vivo and in vitro, and the involvement of the VDR and BAI1-mediated ELMO/Dock180/Rac1 signaling pathway. Furthermore, we will conduct a randomized double-blind placebo-controlled trial to investigate the effect of vitamin D supplementation on the progression of sarcopenia in sarcopenia-affected older people with a vitamin D deficiency. This study seeks to establish a rationale for nutritional therapy by targeting the VDR and BAI1-mediated signaling pathway and providing a new strategy for the prevention or treatment of sarcopenia.

在体内，肌肉干细胞分化为成肌细胞并增殖分化、互相融合形成多核肌纤维，其功能衰减造成肌纤维变细和数量减少是诱发肌肉衰减综合征（肌衰征）的关键机制之一。我国人群普遍缺乏维生素D（VD），而VD受体在正常骨骼肌组织表达。以往，对VD影响成肌细胞增殖和分化研究较多，而对细胞融合的调控机制尚不清楚。我们在前期研究中，发现老年人群VD缺乏与肌衰征密切相关，而肌肉干细胞模型研究表明VD缺乏抑制了成肌细胞的融合功能。本课题拟进一步采用细胞和小鼠模型、肌衰征老年人三个层次探讨VD对细胞融合功能及肌衰征的作用。通过检测VD对细胞和小鼠模型BAI1介导的ELMO/Dock180/Rac1信号通路的作用，阐明VD在细胞融合和肌衰征形成过程中的机制；进而在VD缺乏肌衰征人群中，设计干预试验明确VD补充对肌衰征的改善效果。该研究有助于明确VD缺乏造成肌肉量减少进展为肌衰征的关键机制，为其改善和相关药物研发开辟新途径。

在体内，肌肉干细胞分化为成肌细胞并增殖分化、互相融合形成多核肌纤维，其功能衰减造成肌纤维变细和数量减少是诱发肌肉衰减综合征（肌衰征）的关键机制之一。我国人群普遍缺乏维生素D（VD），而VD受体在正常骨骼肌组织表达。以往，对VD影响成肌细胞增殖和分化研究较多，而对细胞融合的调控机制尚不清楚。我们在前期研究中，发现老年人群VD缺乏与肌衰征密切相关，而肌肉干细胞模型研究表明VD缺乏抑制了成肌细胞的融合功能。本课题进一步采用细胞和小鼠模型、肌衰征老年人三个层次探讨了VD对细胞融合功能及肌衰征的作用。in vitro，我们分析了VD缺乏和补充对来源于肌衰征模型小鼠肌肉干细胞/成肌细胞的作用。结果显示VD3缺乏严重抑制了成肌细胞互相融合形成肌纤维，而VD3干预显著促进了肌纤维融合及其BAI1蛋白的表达水平。另一方面，信号通路分析表明BAI1蛋白下游的细胞内ELMO/Dock180/Rac1信号通路部分参与了活性VD3对成肌细胞分化融合成为肌纤维的作用。in vivo，我们使用肌衰征小鼠模型, 评估了VD3补充对肌衰征表型作用的同时，探讨了VD受体和BAI1介导的ELMO/Dock180/Rac1信号通路是否是其作用的主要机制。结果显示VD3缺乏降低了小鼠握力和转棒上停留时间，而低剂量及高剂量VD3补充显著增加了小鼠握力和转棒上停留时间。此外，结果也显示了VD3缺乏严重抑制了肌损伤后的成肌细胞互相融合进而形成肌纤维。另一方面，VD3补充显著增加了小鼠下肢肌纤维BAI1蛋白的表达水平。我们也观察到了VD3补充对小鼠下肢肌肉质量及肌肉干细胞数量的增加具有一定的作用。可是，信号通路分析表明BAI1蛋白下游的细胞内ELMO/Dock180/Rac1等各活性化蛋白在VD3不同干预组间没有显著差异。最后，我们在VD缺乏肌衰征老年人群中实施双盲随机化干预试验，以验证VD3补充对肌衰征表型的改善作用。结果显示VD3干预明显增加了老年人群血清中25(OH)VD、25(OH)VD2和25(OH)VD3的浓度。与此同时，VD3干预也抑制了步行速度的下降。该研究成果不仅有助于明确VD缺乏造成肌肉量减少进展为肌衰征的关键机制，也为其改善和相关药物研发开辟新途径。