Abstract
BACKGROUND: Bone loss is a significant health concern during spaceflight and mechanical unloading. Simulated microgravity (SMG) disrupts bone homeostasis by inhibiting osteoblast proliferation and differentiation while promoting apoptosis. Although these functional effects have been reported, the underlying mechanisms remain unclear. Mitochondrial quality control, particularly mitophagy involving the PINK1/Parkin pathway, may play a key role. This study aimed to investigate the relationship between osteogenic dysfunction and mitochondrial damage under SMG conditions and preliminarily validate the potential link using the small molecule probe icariin (ICA). METHODS: An SMG model was established using a rotary cell culture system. Cell proliferation was assessed by CCK-8 assay, apoptosis was analyzed via flow cytometry, and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and Alizarin Red staining. Expression levels of relevant genes and proteins were measured by qPCR and Western blot. Mitochondrial function was assessed through ATP content, reactive oxygen species (ROS) levels, JC-1 staining for mitochondrial membrane potential, and transmission electron microscopy (TEM) for ultrastructural observation. Additionally, cells were treated with the mitochondrial function-related small molecule icariin (ICA) to observe its regulatory effects on mitophagy markers (PINK1, Parkin, p62, LC3B) expression and osteogenic function. RESULTS: SMG significantly inhibited osteoblast proliferation and differentiation and induced apoptosis. These changes were accompanied by impaired mitochondrial function and downregulated expression of mitophagy-related genes. TEM revealed mitochondrial swelling and disrupted cristae structure. Treatment with ICA partially restored mitochondrial function and mitophagy marker expression, along with improved expression of osteogenic markers and cell viability. CONCLUSIONS: SMG induces osteogenic dysfunction, mitochondrial damage, and downregulation of mitophagy-related gene expression. The results suggest that impaired mitophagy may be a key mechanism underlying unloading-induced bone loss, and ICA, as a small molecule modulator, holds potential as a therapeutic intervention. CLINICAL TRIAL NUMBER: Not applicable.