Simulated microgravity induces a time-dependent shift from autophagy to apoptosis in osteoblasts, validated by TSPO, ATG12, and BNIP3L expression in vivo.

Emerging evidence indicates that microgravity-induced osteoblast dysfunction is a critical contributor to spaceflight-associated bone loss. This study investigated the temporal dynamics of autophagy-apoptosis crosstalk in MC3T3-E1 osteoblasts under a rotary cell culture system (RCCS)-simulated microgravity. Crucially, time-course analysis (1, 3, 5, 10 days) revealed a biphasic autophagic response: initial enhancement of autophagic flux (LC3-II/Beclin-1 upregulation, p62 degradation) at day 3, exerting cytoprotective effects with reduced apoptosis, subsequently shifting to peak autophagy inhibition at day 5 concomitant with marked apoptosis activation (cleaved caspase-3 elevation) and mitochondrial dysfunction. By day 10, extensive cellular fragmentation dominated. Integrated proteomics identified TSPO, ATG12, and BNip3L as important mediators of this phenotypic switch. Murine hindlimb unloading experiments validated the upregulation of these proteins in bone tissue via Western blot and immunohistochemistry . We hypothesize that Early microgravity exposure triggers compensatory autophagy via ATG12-mediated vesicle expansion, whereas sustained stress is accompanied by TSOPO-associated ROS accumulation and BNip3L-linked alteration in autophagy-related pathways, contributing to apoptosis. Our finding suggests a biphasic temporal pattern in autophagy-apoptosis remodeling under simulated microgravity, highlighting potential time-sensitive windows for future therapeutic targets for spaceflight-associated osteopenia.